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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK code Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV210151 1/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hiPSC (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
95
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin2
Not detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 2.23E7 +- 1.35E7
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 2/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CPC (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin2/ TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 8.27E6 +- 3.53E6
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 3/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMi (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin2/ TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 2.95E7 +- 1.19E7
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 4/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 100%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD63/ Flotillin2
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.08
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMm (IMR90)-4
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ Flotillin2
Not detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: RNA analysis
RNA analysis
Type
RNAsequencing
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250 nm
EV concentration
Yes
Particle yield
number of particles per million cell per 24h;Yes, other: 4.10E7 +- 9.75E6
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 5/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
89% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hiPSC (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
90
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 1.99E7 +- 1.99E6
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 6/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
89% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CPC (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 6.43E06 +- 5.40E05
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 7/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
89% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMi (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 3.49E07 +- 2.85E06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210151 8/8 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Louro, Ana Filipa 2022 89%

Study summary

Full title
All authors
Ana F Louro, Marta A Paiva, Marta R Oliveira, Katharina A Kasper, Paula M Alves, Patrícia Gomes-Alves, Margarida Serra
Journal
Advanced Science
Abstract
Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, i (show more...)Extracellular vesicles (EV) are an attractive therapy to boost cardiac regeneration. Nevertheless, identification of native EV and corresponding cell platform(s) suitable for therapeutic application, is still a challenge. Here, EV are isolated from key stages of the human induced pluripotent stem cell-cardiomyocyte (hiPSC-CM) differentiation and maturation, i.e., from hiPSC (hiPSC-EV), cardiac progenitors, immature and mature cardiomyocytes, with the aim of identifying a promising cell biofactory for EV production, and pinpoint the genetic signatures of bioactive EV. EV secreted by hiPSC and cardiac derivatives show a typical size distribution profile and the expression of specific EV markers. Bioactivity assays show increased tube formation and migration in HUVEC treated with hiPSC-EV compared to EV from committed cell populations. hiPSC-EV also significantly increase cell cycle activity of hiPSC-CM. Global miRNA expression profiles, obtained by small RNA-seq analysis, corroborate an EV-miRNA pattern indicative of stem cell to cardiomyocyte specification, confirming that hiPSC-EV are enriched in pluripotency-associated miRNA with higher in vitro pro-angiogenic and pro-proliferative properties. In particular, a stemness maintenance miRNA cluster upregulated in hiPSC-EV targets the PTEN/PI3K/AKT pathway, involved in cell proliferation and survival. Overall, the findings validate hiPSC as cell biofactories for EV production for cardiac regenerative applications. (hide)
EV-METRIC
89% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Filtration
Protein markers
EV: TSG101
non-EV: Argonaute2
Proteomics
no
EV density (g/ml)
1.083
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
CMm (DF19-9-11T.H)
EV-harvesting Medium
Serum free medium
Cell viability (%)
85
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
180
Pelleting: rotor type
SW 28
Pelleting: speed (g)
110000
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
16.5
Sample volume (mL)
0.8
Orientation
Bottom-up
Rotor type
SW 28.1
Speed (g)
110000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
None
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101
Not detected contaminants
Argonaute2
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-250
EV concentration
Yes
Particle yield
number of particles per million cells per 24h;Yes, other: 2.13E07 +- 2.25E06
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210199 1/1 Homo sapiens Urine (d)(U)C
Filtration
Correll, Vanessa 2022 78%

Study summary

Full title
All authors
Vanessa L. Correll, Joseph J. Otto, Cristina M. Risi, Brian P. Main, Paul C. Boutros, Thomas Kislinger, Vitold E. Galkin, Julius O. Nyalwidhe, O. John Semmes, Lifang Yang
Journal
J Extracell Vesicles
Abstract
The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient (show more...)The isolation and subsequent molecular analysis of extracellular vesicles (EVs) derived from patient samples is a widely used strategy to understand vesicle biology and to facilitate biomarker discovery. Expressed prostatic secretions in urine are a tumor proximal fluid that has received significant attention as a source of potential prostate cancer (PCa) biomarkers for use in liquid biopsy protocols. Standard EV isolation methods like differential ultracentrifugation (dUC) co-isolate protein contaminants that mask lower-abundance proteins in typical mass spectrometry (MS) protocols. Further complicating the analysis of expressed prostatic secretions, uromodulin, also known as Tamm-Horsfall protein (THP), is present at high concentrations in urine. THP can form polymers that entrap EVs during purification, reducing yield. Disruption of THP polymer networks with dithiothreitol (DTT) can release trapped EVs, but smaller THP fibres co-isolate with EVs during subsequent ultracentrifugation. To resolve these challenges, we describe here a dUC method that incorporates THP polymer reduction and alkaline washing to improve EV isolation and deplete both THP and other common protein contaminants. When applied to human expressed prostatic secretions in urine, we achieved relative enrichment of known prostate and prostate cancer-associated EV-resident proteins. Our approach provides a promising strategy for global proteomic analyses of urinary EVs. (hide)
EV-METRIC
78% (97th 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
Urine
Sample origin
prostate cancer
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: TSG101/ CD9
non-EV: Calnexin/ Tamm-Horsfall protein
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods/Biomarker/New methodological development
Sample
Species
Homo sapiens
Sample Type
Urine
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
175000
Wash: volume per pellet (ml)
12.5
Wash: time (min)
130
Wash: Rotor Type
SW 40 Ti
Wash: speed (g)
175000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
per 1E10 particles: 1.16
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ TSG101
Detected contaminants
Tamm-Horsfall protein
Not detected contaminants
Calnexin
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
145.9
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.20E+09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210187 1/5 Homo sapiens Cell culture supernatant (d)(U)C Swatler, Julian 2022 78%

Study summary

Full title
All authors
Julian Swatler, Laura Turos-Korgul, Marta Brewinska-Olchowik, Sara De Biasi, Wioleta Dudka, Bac Viet Le, Agata Kominek, Salwador Cyranowski, Paulina Pilanc, Elyas Mohammadi, Dominik Cysewski, Ewa Kozlowska, Wioleta Grabowska-Pyrzewicz, Urszula Wojda, Grzegorz W Basak, Jakub Mieczkowski, Tomasz Skorski 10 , Andrea Cossarizza 11 , Katarzyna Piwocka
Journal
Blood advances
Abstract
Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosupp (show more...)Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosuppression by expanding pro-leukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector, pro-leukemic Tregs. Using mouse model of CML-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, IL21R and included two distinct, effector Treg (eTreg) subsets - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive regulatory T cells and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms. (hide)
EV-METRIC
78% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ Alix/ CD63/ CD81
non-EV: APOA1/ GM130
Proteomics
yes
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
K562
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
98
Cell count
3.00E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
60
Wash: time (min)
90
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100,000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD63/ TSG101/ Alix/ CD81
Not detected contaminants
APOA1/ GM130
Proteomics database
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
94
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 3.00E+07
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210002 1/3 Mus musculus dissociated tissues (d)(U)C
Delcorte, Ophélie 2022 78%

Study summary

Full title
All authors
Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux
Journal
Biomediscines
Abstract
Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide)
EV-METRIC
78% (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. 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
dissociated tissues
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
No extra separation steps
Protein markers
EV: Alix/ CD63/ Flotillin1/ CD9/ CD81
non-EV: Calnexin/ PDI
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
dissociated tissues
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 80 Ti
Pelleting: speed (g)
150000
Wash: volume per pellet (ml)
7
Wash: time (min)
90
Wash: Rotor Type
Type 80 Ti
Wash: speed (g)
150000
Other
Name other separation method
No extra separation steps
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ Alix/ CD81
Not detected contaminants
Calnexin/ PDI
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;RNA sequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
Before
RNAse type
RNase A
RNAse concentration
0,01
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
Particle yield
Per dissociated thyroid;Yes, other: 1,50E+09
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Close-up, Wide-field
EV220003 1/2 Homo sapiens Blood plasma DG
SEC (non-commercial)
Karimi, Nasibeh 2022 75%

Study summary

Full title
All authors
Nasibeh Karimi, Razieh Dalirfardouei, Tomás Dias, Jan Lötvall, Cecilia Lässer
Journal
J Extracell Vesicles
Abstract
Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the developme (show more...)Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail. (hide)
EV-METRIC
75% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin
non-EV: Calnexin
Proteomics
no
EV density (g/ml)
1.06-1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
12 ml
Sample volume (mL)
6 ml
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
178,000
Duration (min)
180
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
27.8
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin
Not detected contaminants
Calnexin
Detected EV-associated proteins
CD9/ CD63/ CD81/ CD41a
Detected EV-associated proteins
CD9/ CD63/ CD81/ CD41a
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
as total number of particles in pooled SEC fractions from 6 mL of sample: 2.90e+9
Report type
Size range/distribution
Report size
50-70
EV-concentration
No
EV210383 1/2 Mus musculus Serum ExoQuick Mkrtchian, Soiuren 2022 75%

Study summary

Full title
All authors
Souren Mkrtchian, Anette Ebberyd, Rosanne E. Veerman, María Méndez-Lago, Susanne Gabrielsson, Lars I. Eriksson, and Marta Gómez-Galán
Journal
Front Immunol
Abstract
Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling respon (show more...)Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain. (hide)
EV-METRIC
75% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
ExoQuick
Protein markers
EV: CD63/ CD81/ HSP70
non-EV: Albumin/ Argonaute?2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Serum
Separation Method
Commercial kit
ExoQuick
Other
Name other separation method
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
0.75
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ CD81/ HSP70
Not detected contaminants
Albumin
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210383 2/2 Mus musculus Serum IAF Mkrtchian, Soiuren 2022 75%

Study summary

Full title
All authors
Souren Mkrtchian, Anette Ebberyd, Rosanne E. Veerman, María Méndez-Lago, Susanne Gabrielsson, Lars I. Eriksson, and Marta Gómez-Galán
Journal
Front Immunol
Abstract
Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling respon (show more...)Surgical interventions rapidly trigger a cascade of molecular, cellular, and neural signaling responses that ultimately reach remote organs, including the brain. Using a mouse model of orthopedic surgery, we have previously demonstrated hippocampal metabolic, structural, and functional changes associated with cognitive impairment. However, the nature of the underlying signals responsible for such periphery-to-brain communication remains hitherto elusive. Here we present the first exploratory study that tests the hypothesis of extracellular vesicles (EVs) as potential mediators carrying information from the injured tissue to the distal organs including the brain. The primary goal was to investigate whether the cargo of circulating EVs after surgery can undergo quantitative changes that could potentially trigger phenotypic modifications in the target tissues. EVs were isolated from the serum of the mice subjected to a tibia surgery after 6, 24, and 72 h, and the proteome and miRNAome were investigated using mass spectrometry and RNA-seq approaches. We found substantial differential expression of proteins and miRNAs starting at 6 h post-surgery and peaking at 24 h. Interestingly, one of the up-regulated proteins at 24 h was α-synuclein, a pathogenic hallmark of certain neurodegenerative syndromes. Analysis of miRNA target mRNA and corresponding biological pathways indicate the potential of post-surgery EVs to modify the extracellular matrix of the recipient cells and regulate metabolic processes including fatty acid metabolism. We conclude that surgery alters the cargo of circulating EVs in the blood, and our results suggest EVs as potential systemic signal carriers mediating remote effects of surgery on the brain. (hide)
EV-METRIC
75% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Immunoaffinity capture (non-commercial)
Protein markers
EV: CD63/ CD81/ HSP70
non-EV: Albumin/ Argonaute?2/ Calreticulin/ GM130/ PMP70/ Prohibitin/ Tamm-Horsfall protein
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Serum
Separation Method
Immunoaffinity capture
Selected surface protein(s)
CD9/ CD63/ CD81
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
0.75
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ CD81/ HSP70
Detected contaminants
Albumin
Proteomics database
Yes
Characterization: RNA analysis
RNA analysis
Type
(RT)?(q)PCR/ RNA?sequencing
Database
NGI
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
100
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210237 1/2 Homo sapiens Blood plasma qEV Gelibter S 2022 75%

Study summary

Full title
All authors
Gelibter S, Marostica G, Mandelli A, Siciliani S, Podini P, Finardi A, Furlan R
Journal
J Extracell Vesicles
Abstract
Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. Wh (show more...)Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While -80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of -80°C storage and freeze-thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment/ flow cytometry for the analysis of two single fluorescent-tagged EVs populations (GFP and mCherry), mixed before preservation. We found that -80°C storage reduces EVs concentration and sample purity in a time-dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size-charge plots. None of the tested conditions prevented the observed effects. Freeze-thaw cycles lead to an EVs reduction after the first cycle and to a cycle-dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double-positive EVs (GFP -mCherry ). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non-archival, samples in majority of cases. (hide)
EV-METRIC
75% (97th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
qEV
Protein markers
EV: Alix/ Flotillin-1/ Lamp1/ ANXA1
non-EV: GM130/ ApoE/ H3
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
particles per milliliter of starting sample
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ Flotillin-1/ Lamp1/ ANXA1
Not detected contaminants
GM130/ ApoE/ H3
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
50-350
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.00e+11
EM
EM-type
Immuno-EM
EM protein
CD63
Image type
Wide-field
EV200029 1/2 Bos taurus Bovine embryo culture medium qEV Pavani KC 2022 75%

Study summary

Full title
All authors
Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K
Journal
Proc Natl Acad Sci U S A
Abstract
SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (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. 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
Bovine embryo culture medium
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Commercial method
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: AGO2/ APOA1
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Bos taurus
Sample Type
Bovine embryo culture medium
Separation Method
Commercial kit
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
CD9/ CD63/ TSG101
Detected contaminants
APOA1
Not detected contaminants
AGO2
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
Before
RNAse type
RNase A
RNAse concentration
10
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
94.5±1.7 E08
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
EV200029 2/2 Bos taurus Bovine embryo culture medium qEV Pavani KC 2022 75%

Study summary

Full title
All authors
Pavani KC, Meese T, Pascottini OB, Guan X, Lin X, Peelman L, Hamacher J, Van Nieuwerburgh F, Deforce D, Boel A, Heindryckx B, Tilleman K, Van Soom A, Gadella BM, Hendrix A, Smits K
Journal
Proc Natl Acad Sci U S A
Abstract
SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less l (show more...)SignificanceHatching from the zona pellucida is a prerequisite for embryo implantation and is less likely to occur in vitro for reasons unknown. Extracellular vesicles (EVs) are secreted by the embryo into the culture medium. Yet the role that embryonic EVs and their cargo microRNAs (miRNAs) play in blastocyst hatching has not been elucidated, partially due to the difficulties of isolating them from low amounts of culture medium. Here, we optimized EV-miRNA isolation from medium conditioned by individually cultured bovine embryos and subsequently showed that miR-378a-3p, which was up-regulated in EVs secreted by blastocysts, plays a crucial role in promoting blastocyst hatching. This demonstrates the regulatory effect of miR-378-3p on hatching, which is an established embryo quality parameter linked with implantation. (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. 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
Bovine embryo culture medium
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Commercial method
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: AGO2/ APOA1
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Bos taurus
Sample Type
Bovine embryo culture medium
Separation Method
Commercial kit
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
CD9/ CD63/ TSG101
Detected contaminants
APOA1
Not detected contaminants
AGO2
Characterization: RNA analysis
RNA analysis
Type
RNA sequencing
Database
No
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
Before
RNAse type
RNase A
RNAse concentration
10
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
76.5±8.2 E08
EM
EM-type
Transmission-EM/ Cryo-EM
Image type
Close-up, Wide-field
EV210187 3/5 Homo sapiens Cell culture supernatant (d)(U)C Swatler, Julian 2022 67%

Study summary

Full title
All authors
Julian Swatler, Laura Turos-Korgul, Marta Brewinska-Olchowik, Sara De Biasi, Wioleta Dudka, Bac Viet Le, Agata Kominek, Salwador Cyranowski, Paulina Pilanc, Elyas Mohammadi, Dominik Cysewski, Ewa Kozlowska, Wioleta Grabowska-Pyrzewicz, Urszula Wojda, Grzegorz W Basak, Jakub Mieczkowski, Tomasz Skorski 10 , Andrea Cossarizza 11 , Katarzyna Piwocka
Journal
Blood advances
Abstract
Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosupp (show more...)Chronic and acute myeloid leukemia (CML, AML) evade immune system surveillance and induce immunosuppression by expanding pro-leukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector, pro-leukemic Tregs. Using mouse model of CML-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, IL21R and included two distinct, effector Treg (eTreg) subsets - CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive regulatory T cells and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101
non-EV: APOA1/ GM130
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MOLM-14
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
98
Cell count
2.80E+08
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
60
Wash: time (min)
90
Wash: Rotor Type
Type 45 Ti
Wash: speed (g)
100,000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
TSG101
Not detected contaminants
APOA1/ GM130
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
95
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 5.00E+07
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210168 1/3 Homo sapiens Blood plasma qEV
IAF
Newman, Lauren 2022 67%

Study summary

Full title
All authors
Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland
Journal
biomedicines
Abstract
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Commercial method
Immunoaffinity capture (non-commercial)
Protein markers
EV: None
non-EV: albumin/ calnexin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Commercial kit
qEV
Immunoaffinity capture
Selected surface protein(s)
ASGR1
Characterization: Protein analysis
Protein Concentration Method
microBCA
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
102.9
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.17E+11
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210168 2/3 Homo sapiens Blood plasma qEV
IAF
Newman, Lauren 2022 67%

Study summary

Full title
All authors
Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland
Journal
biomedicines
Abstract
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
NAFL
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
Commercial method
Immunoaffinity capture (non-commercial)
Protein markers
EV: None
non-EV: albumin/ calnexin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Commercial kit
qEV
Immunoaffinity capture
Selected surface protein(s)
ASGR1
Characterization: Protein analysis
Protein Concentration Method
microBCA
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
113.3
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.34E+11
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210168 3/3 Homo sapiens Blood plasma qEV
IAF
Newman, Lauren 2022 67%

Study summary

Full title
All authors
Lauren A Newman, Zivile Useckaite, Jillian Johnson, Michael J Sorich, Ashley M Hopkins, Andrew Rowland
Journal
biomedicines
Abstract
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagn (show more...)Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Definitive diagnosis of the progressive form, non-alcoholic steatohepatitis (NASH), requires liver biopsy, which is highly invasive and unsuited to early disease or tracking changes. Inadequate performance of current minimally invasive tools is a critical barrier to managing NAFLD burden. Altered circulating miRNA profiles show potential for minimally invasive tracking of NAFLD. The selective isolation of the circulating extracellular vesicle subset that originates from hepatocytes presents an important opportunity for improving the performance of miRNA biomarkers of liver disease. The expressions of miR-122, -192, and -128-3p were quantified in total cell-free RNA, global EVs, and liver-specific EVs from control, NAFL, and NASH subjects. In ASGR1+ EVs, each miR biomarker trended positively with disease severity and expression was significantly higher in NASH subjects compared with controls. The c-statistic defining the performance of ASGR1+ EV derived miRNAs was invariably >0.78. This trend was not observed in the alternative sources. This study demonstrates the capacity for liver-specific isolation to transform the performance of EV-derived miRNA biomarkers for NAFLD, robustly distinguishing patients with NAFL and NASH. (hide)
EV-METRIC
67% (95th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
NASH
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
Commercial method
Immunoaffinity capture (non-commercial)
Protein markers
EV: None
non-EV: albumin/ calnexin
Proteomics
yes
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Commercial kit
qEV
Immunoaffinity capture
Selected surface protein(s)
ASGR1
Characterization: Protein analysis
Protein Concentration Method
microBCA
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR
Database
No
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
110.1
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.73E+11
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV210002 2/3 Mus musculus dissociated tissues (d)(U)C
Delcorte, Ophélie 2022 67%

Study summary

Full title
All authors
Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux
Journal
Biomediscines
Abstract
Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide)
EV-METRIC
67% (33rd 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
dissociated tissues
Sample origin
BRAFV600E-induced tissue (Doxycyxline 2*24h)
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
No extra separation steps
Protein markers
EV: Alix/ CD63/ Flotillin1/ CD9/ CD81
non-EV: Calnexin/ PDI
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
dissociated tissues
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 80 Ti
Pelleting: speed (g)
150000
Wash: volume per pellet (ml)
7
Wash: time (min)
90
Wash: Rotor Type
Type 80 Ti
Wash: speed (g)
150000
Other
Name other separation method
No extra separation steps
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ Alix/ CD81
Not detected contaminants
Calnexin/ PDI
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;RNAsequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
Before
RNAse type
RNase A
RNAse concentration
0,01
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
Particle yield
Per dissociated thyroid;Yes, other: 3,30E+09
EM
EM-type
Transmission-EM
Image type
Close-up
EV210002 3/3 Mus musculus dissociated tissues (d)(U)C
Delcorte, Ophélie 2022 67%

Study summary

Full title
All authors
Ophélie Delcorte, Catherine Spourquet, Pascale Lemoine, Jonathan Degosserie, Patrick Van Der Smissen, Nicolas Dauguet, Axelle Loriot, Jeffrey A Knauf, Laurent Gatto, Etienne Marbaix, James A Fagin, Christophe E Pierreux
Journal
Biomediscines
Abstract
Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recur (show more...)Papillary thyroid cancer (PTC) is the most common endocrine malignancy for which diagnosis and recurrences still challenge clinicians. New perspectives to overcome these issues could come from the study of extracellular vesicle (EV) populations and content. Here, we aimed to elucidate the heterogeneity of EVs circulating in the tumor and the changes in their microRNA content during cancer progression. Using a mouse model expressing BRAFV600E, we isolated and characterized EVs from thyroid tissue by ultracentrifugations and elucidated their microRNA content by small RNA sequencing. The cellular origin of EVs was investigated by ExoView and that of deregulated EV-microRNA by qPCR on FACS-sorted cell populations. We found that PTC released more EVs bearing epithelial and immune markers, as compared to the healthy thyroid, so that changes in EV-microRNAs abundance were mainly due to their deregulated expression in thyrocytes. Altogether, our work provides a full description of in vivo-derived EVs produced by, and within, normal and cancerous thyroid. We elucidated the global EV-microRNAs signature, the dynamic loading of microRNAs in EVs upon BRAFV600E induction, and their cellular origin. Finally, we propose that thyroid tumor-derived EV-microRNAs could support the establishment of a permissive immune microenvironment. (hide)
EV-METRIC
67% (33rd 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
dissociated tissues
Sample origin
BRAFV600E-induced tissue (Doxycyxline4*24h)
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
No extra separation steps
Protein markers
EV: Alix/ CD63/ Flotillin1/ CD9/ CD81
non-EV: Calnexin/ PDI
Proteomics
no
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
dissociated tissues
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 80 Ti
Pelleting: speed (g)
150000
Wash: volume per pellet (ml)
7
Wash: time (min)
90
Wash: Rotor Type
Type 80 Ti
Wash: speed (g)
150000
Other
Name other separation method
No extra separation steps
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
Flotillin1/ Alix/ CD9/ CD63/ CD81
Not detected contaminants
Calnexin/ PDI
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;RNAsequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
Before
RNAse type
RNase A
RNAse concentration
0,01
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
150
EV concentration
Yes
Particle yield
Per dissociated thyroid;Yes, other: 1,20E+10
EM
EM-type
Transmission-EM
Image type
Close-up
EV200174 1/2 Bos taurus 1% skim milk (d)(U)C
Sukreet, Sonal 2022 67%

Study summary

Full title
All authors
Sonal Sukreet, Camila Pereira Braga, Thuy T An, Jiri Adamec, Juan Cui, Janos Zempleni
Journal
J Nutr
Abstract
Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumptio (show more...)Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumption of an exosome and RNA-depleted (ERD) diet elicited phenotypes compared to controls fed an exosome and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and non-ultrasonicated milk, respectively, for casein in the AIN-93G formulation. Objective: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. Methods: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation (USE); controls were not ultrasonicated (NSE). Exosome count, size and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids and proteins were analyzed by RNA-sequencing and mass spectrometry. Intestinal transport, bioavailability and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells and C57BL/6J mice (n = 3; age 6 - 8 weeks). Results: The exosome count was 76 ± 22% lower in USE compared to NSE (P < 0.05). Ultrasonication caused a degradation of up to 100% of microRNAs. USE and NSE contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected total of 525 and 484 proteins in USE and NSE. The uptake of USEs decreased by 46 ± 30% and 40 ± 27% compared to NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). Conclusions: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells and C57BL/6J mice and causes a near-complete degradation of microRNA cargos. (hide)
EV-METRIC
67% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
1% skim milk
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
No extra separation steps
Protein markers
EV: TSG101/ Alix/ CD63/ CD9/ CD81
non-EV: Beta-integrin/ lactalbumin/ Histone-H3
Proteomics
yes
Show all info
Study aim
Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Bos taurus
Sample Type
1% skim milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Fiberlite-F37L-8x100 rotor
Pelleting: speed (g)
120,000
Wash: volume per pellet (ml)
30 mL
Wash: time (min)
90
Wash: Rotor Type
Fiberlite-F37L-8x100 rotor
Wash: speed (g)
120,000
Other
Name other separation method
No extra separation steps
Characterization: Protein analysis
Protein Concentration Method
BCA;Qubit
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ TSG101/ Alix/ CD81
Detected contaminants
lactalbumin
Not detected contaminants
Beta-integrin/ Histone-H3
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;RNA sequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
117+/-44
EV concentration
Yes
Particle yield
Yes, per milliliter of starting sample 5.90E+11
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Close-up, Wide-field
EV200174 2/2 Bos taurus 1% skim milk (d)(U)C Sukreet, Sonal 2022 67%

Study summary

Full title
All authors
Sonal Sukreet, Camila Pereira Braga, Thuy T An, Jiri Adamec, Juan Cui, Janos Zempleni
Journal
J Nutr
Abstract
Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumptio (show more...)Background: Bovine milk exosomes (BMEs) harbor regulatory proteins, lipids and microRNAs. Consumption of an exosome and RNA-depleted (ERD) diet elicited phenotypes compared to controls fed an exosome and RNA-sufficient (ERS) diet in mice. All other ingredients were identical in the diets. ERD and ERS diets were prepared by substituting ultrasonicated and non-ultrasonicated milk, respectively, for casein in the AIN-93G formulation. Objective: The objective of this study was to assess the effect of ultrasonication of milk on exosome content and bioavailability, and cargo content. Methods: Bovine milk was ultrasonicated and exosomes were isolated by ultracentrifugation (USE); controls were not ultrasonicated (NSE). Exosome count, size and morphology were assessed using a nanoparticle tracker and electron microscopy. RNAs, lipids and proteins were analyzed by RNA-sequencing and mass spectrometry. Intestinal transport, bioavailability and distribution were measured by using fluorophore-labeled USEs and NSEs in Caco-2 cells, FHs 74 Int cells and C57BL/6J mice (n = 3; age 6 - 8 weeks). Results: The exosome count was 76 ± 22% lower in USE compared to NSE (P < 0.05). Ultrasonication caused a degradation of up to 100% of microRNAs. USE and NSE contained 145 and 332 unique lipid signatures, respectively (P < 0.05). We detected total of 525 and 484 proteins in USE and NSE. The uptake of USEs decreased by 46 ± 30% and 40 ± 27% compared to NSEs in Caco-2 and FHs 74 Int cells, respectively (P < 0.05). The hepatic accumulation of USEs was 48% ± 28% lower than the accumulation of NSEs in mice (P < 0.05). Conclusions: Ultrasonication of milk depletes bioavailable BMEs in studies of Caco-2 cells, FHs 74 Int cells and C57BL/6J mice and causes a near-complete degradation of microRNA cargos. (hide)
EV-METRIC
67% (50th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
1% skim milk
Sample origin
Sonicated milk
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Protein markers
EV: CD81/ Alix/ TSG101/ CD63/ CD9
non-EV: Beta-integrin/ lactalbumin/ Histone H3
Proteomics
yes
Show all info
Study aim
Function/New methodological development/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Bos taurus
Sample Type
1% skim milk
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Fiberlite-F37L-8x100 rotor
Pelleting: speed (g)
120,000
Wash: volume per pellet (ml)
30 mL
Wash: time (min)
90
Wash: Rotor Type
Fiberlite-F37L-8x100 rotor
Wash: speed (g)
120,000
Characterization: Protein analysis
Protein Concentration Method
BCA;Qubit
Western Blot
Antibody details provided?
No
Detected EV-associated proteins
CD9/ CD63/ TSG101
Not detected EV-associated proteins
CD81/ Alix
Detected contaminants
lactalbumin
Not detected contaminants
Beta-integrin/ Histone H3
Proteomics database
No
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR;RNAsequencing
Database
Yes
Proteinase treatment
No
RNAse treatment
No
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
218+/-99
EV concentration
Yes
Particle yield
Yes, per milliliter of starting sample 1.40E+11
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Close-up, Wide-field
EV200112 1/2 Homo sapiens Cell culture supernatant (d)(U)C
Giraud, Romain 2022 67%

Study summary

Full title
All authors
Romain Giraud, Anaïs Moyon, Stéphanie Simoncini, Anne-Claire Duchez, Vincent Nail, Corinne Chareyre, Ahlem Bouhlel, Laure Balasse, Samantha Fernandez, Loris Vallier, Guillaume Hache, Florence Sabatier, Françoise Dignat-George, Romaric Lacroix, Benjamin Guillet, Philippe Garrigue
Journal
Pharmaceutics
Abstract
Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as b (show more...)Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. Methods: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. Results: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. Conclusions: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
No extra separation steps
Protein markers
EV: CD51/ CD63/ CD81/ CD31/ CD146/ caveolin/ ICAM1/ integrin beta 3
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
JA-30.50
Pelleting: speed (g)
70000
Wash: volume per pellet (ml)
30
Wash: time (min)
90
Wash: Rotor Type
JA-30.50
Wash: speed (g)
70000
Other
Name other separation method
No extra separation steps
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ CD51/ caveolin/ integrin-beta3/ CD81
Not detected contaminants
Albumin
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD146/ ICAM1/ CD31
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
434.5
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
434
EV200112 2/2 Homo sapiens Cell culture supernatant (d)(U)C
qEV
Giraud, Romain 2022 67%

Study summary

Full title
All authors
Romain Giraud, Anaïs Moyon, Stéphanie Simoncini, Anne-Claire Duchez, Vincent Nail, Corinne Chareyre, Ahlem Bouhlel, Laure Balasse, Samantha Fernandez, Loris Vallier, Guillaume Hache, Florence Sabatier, Françoise Dignat-George, Romaric Lacroix, Benjamin Guillet, Philippe Garrigue
Journal
Pharmaceutics
Abstract
Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as b (show more...)Microvesicles, so-called endothelial large extracellular vesicles (LEVs), are of great interest as biological markers and cell-free biotherapies in cardiovascular and oncologic diseases. However, their therapeutic perspectives remain limited due to the lack of reliable data regarding their systemic biodistribution after intravenous administration. Methods: Applied to a mouse model of peripheral ischemia, radiolabeled endothelial LEVs were tracked and their in vivo whole-body distribution was quantified by microSPECT/CT imaging. Hindlimb perfusion was followed by LASER Doppler and motility impairment function was evaluated up to day 28 post-ischemia. Results: Early and specific homing of LEVs to ischemic hind limbs was quantified on the day of ischemia and positively correlated with reperfusion intensity at a later stage on day 28 after ischemia, associated with an improved motility function. Conclusions: This concept is a major asset for investigating the biodistribution of LEVs issued from other cell types, including cancer, thus partly contributing to better knowledge and understanding of their fate after injection. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Commercial method
Protein markers
EV: beta 3 integrin/ CD51/ CD63/ CD81/ CD31/ CD146/ caveolin/ ICAM1
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HUVEC
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 50,000 g and 100,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
JA-30.50
Pelleting: speed (g)
70000
Wash: volume per pellet (ml)
30
Wash: time (min)
90
Wash: Rotor Type
JA-30.50
Wash: speed (g)
70000
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ CD51/ caveolin/ integrin-beta3/ CD81
Not detected contaminants
Albumin
Flow cytometry aspecific beads
Antibody details provided?
No
Detected EV-associated proteins
CD31/ CD146/ ICAM1
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Size range/distribution
Reported size (nm)
434.5
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
407
EV200070 1/2 Homo sapiens Cell culture supernatant (d)(U)C Russell, Ashley 2022 67%

Study summary

Full title
All authors
Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication
Journal
Toxicol Lett.
Abstract
Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contribu (show more...)Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: CD81/ TSG101/ CD63/ Syntenin
non-EV: Calnexin/ GM130
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary CD14+ cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
AH-629 (36 ml)
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63
Not detected EV-associated proteins
CD81/ Syntenin/ TSG101
Detected contaminants
Calnexin/ GM130
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
245.6 nm
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 6.89E+09
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200070 2/2 Homo sapiens Cell culture supernatant (d)(U)C Russell, Ashley 2022 67%

Study summary

Full title
All authors
Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication
Journal
Toxicol Lett.
Abstract
Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contribu (show more...)Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
cells exposed to cigarette smoke extract
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD81/ CD63/ Syntenin
non-EV: Calnexin/ GM130
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary CD14+ cells
EV-harvesting Medium
Serum free medium
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
AH-629 (36 ml)
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ Syntenin/ CD81
Not detected EV-associated proteins
TSG101
Detected contaminants
Calnexin/ GM130
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
252.45 nm
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 8.51E+10
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220003 2/2 Homo sapiens Serum DG
SEC (non-commercial)
Karimi, Nasibeh 2022 63%

Study summary

Full title
All authors
Nasibeh Karimi, Razieh Dalirfardouei, Tomás Dias, Jan Lötvall, Cecilia Lässer
Journal
J Extracell Vesicles
Abstract
Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the developme (show more...)Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail. (hide)
EV-METRIC
63% (95th 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
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.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin
non-EV: None
Proteomics
no
EV density (g/ml)
1.06-1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
12 ml
Sample volume (mL)
6 ml
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
178,000
Duration (min)
180
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
26.7
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected EV-associated proteins
Flotillin-1/ CD41a/ P-selectin
Detected EV-associated proteins
CD9/ CD63/ CD81/ CD41a
Detected EV-associated proteins
CD9/ CD63/ CD81/ CD41a
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
as total number of particles in pooled SEC fractions from 6 mL of sample: 2.56e+9
Report type
Size range/distribution
Report size
50-70
EV-concentration
No
EV210214 1/1 Homo sapiens Cell culture supernatant (d)(U)C
UF
SEC (non-commercial)
Bonifay, Amandine 2022 57%

Study summary

Full title
All authors
Amandine Bonifay, Stéphane Robert, Belinda Champagne, Paul‐Rémi Petit, Aude Eugène, Corinne Chareyre, Anne‐Claire Duchez, Mélanie Vélier, Shirley Fritz, Loris Vallier, Romaric Lacroix, Françoise Dignat‐George
Journal
J Extracell Vesicles
Abstract
Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcel (show more...)Newly recognized polymorphonuclear neutrophil (PMNs) functions include the ability to release subcellular mediators such as neutrophil‐derived extracellular vesicles (NDEVs) involved in immune and thrombo‐inflammatory responses. Elevation of their plasmatic level has been reported in a variety of infectious and cardiovascular disorders, but the clinical use of this potential biomarker is hampered by methodological issues. Although flow cytometry (FCM) is currently used to detect NDEVs in the plasma of patients, an extensive characterization of NDEVs has never been done. Moreover, their detection remains challenging because of their small size and low antigen density. Therefore, the objective of the present study was first to establish a surface antigenic signature of NDEVs detectable by FCM and therefore to improve their detection in biological fluids by developing a strategy allowing to overcome their low fluorescent signal and reduce the background noise. By testing a large panel of 54 antibody specificities already reported to be positive on PMNs, we identified a profile of 15 membrane protein markers, including 4 (CD157, CD24, CD65 and CD66c) never described on NDEVs. Among them, CD15, CD66b and CD66c were identified as the most sensitive and specific markers to detect NDEVs by FCM. Using this antigenic signature, we developed a new strategy combining the three best antibodies in a cocktail and reducing the background noise by size exclusion chromatography (SEC). This strategy allowed a significant improvement in NDEVs enumeration in plasma from sepsis patients and made it feasible to efficiently sort NDEVs from COVID‐19 patients. Altogether, this work opens the door to a more valuable measurement of NDEVs as a potential biomarker in clinical practice. A similar strategy could also be applied to improve detection by FCM of other rare subpopulations of EVs generated by tissues with limited access, such as vascular endothelium, cancer cells or placenta. (hide)
EV-METRIC
57% (88th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
large 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
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD63/ GAPDH/ Integrin-beta3-subunit/ CD15/ CD66b/ CD44/ CD11c
non-EV: Albumin
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
primary neutrophils
EV-harvesting Medium
Serum-containing medium
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
0.5
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
CD63/ GAPDH/ Integrin-beta3-subunit
Detected contaminants
Albumin
Flow cytometry
Type of Flow cytometry
Cytoflex S
Calibration bead size
0.1 0.16 0.22 0.24 0.3 0.5 0.9
Antibody details provided?
Yes
Detected EV-associated proteins
CD15/ CD66b
Not detected EV-associated proteins
CD44/ CD11c
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Median
Reported size (nm)
191
Particle analysis: flow cytometry
Flow cytometer type
Cytoflex S
Hardware adjustment
Calibration bead size
0.1 0.16 0.22 0.24 0.3 0.5 0.9
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
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% (60th 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
Characterization: Particle analysis
None
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% (60th 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
Characterization: Particle analysis
None
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% (60th 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% (60th 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% (60th 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% (60th 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