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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV200036 5/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
qEV
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
human skin primary fibroblasts
Sample origin
Progeria patients
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
qEV
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Progeria patients
EV-producing cells
human skin primary fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 7/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
qEV
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
primary human foreskin fibroblasts
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
qEV
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 9/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
qEV
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
primary human foreskin fibroblasts
Sample origin
iRas
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
qEV
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
iRas
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 11/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
qEV
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
primary human foreskin fibroblasts
Sample origin
iRas+GSTM2
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
qEV
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
iRas+GSTM2
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 13/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
qEV
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
primary human foreskin fibroblasts
Sample origin
iC+GSTM2
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
qEV
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
iC+GSTM2
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200036 15/16 Homo sapiens Cell culture supernatant DG
(d)(U)C
qEV
Juan Antonio Fafián-Labora 2020 56%

Study summary

Full title
All authors
Juan Antonio Fafián-Labora, Jose Antonio Rodríguez-Navarro, Ana O'Loghlen
Journal
Cell metab
Abstract
Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, includin (show more...)Aging is a process of cellular and tissue dysfunction characterized by different hallmarks, including cellular senescence. However, there is proof that certain features of aging and senescence can be ameliorated. Here, we provide evidence that small extracellular vesicles (sEVs) isolated from primary fibroblasts of young human donors ameliorate certain biomarkers of senescence in cells derived from old and Hutchinson-Gilford progeria syndrome donors. Importantly, sEVs from young cells ameliorate senescence in a variety of tissues in old mice. Mechanistically, we identified sEVs to have intrinsic glutathione-S-transferase activity partially due to the high levels of expression of the glutathione-related protein (GSTM2). Transfection of recombinant GSTM2 into sEVs derived from old fibroblasts restores their antioxidant capacity. sEVs increase the levels of reduced glutathione and decrease oxidative stress and lipid peroxidation both in vivo and in vitro. Altogether, our data provide an indication of the potential of sEVs as regenerative therapy in aging. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
primary human foreskin fibroblasts
Sample origin
mCherry-CD63
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
qEV
Protein markers
EV: Alix/ GSTM2
non-EV: None
Proteomics
no
EV density (g/ml)
1.074-1.106
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
mCherry-CD63
EV-producing cells
primary human foreskin fibroblasts
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >= 100,000g
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
15
Wash: time (min)
80
Wash: Rotor Type
T-865
Wash: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
5.5
Sample volume (mL)
1.5
Orientation
Bottom-up
Rotor type
T-865
Speed (g)
100000
Duration (min)
720
Fraction volume (mL)
0.7
Fraction processing
Centrifugation
Pelleting: volume per fraction
15
Pelleting: duration (min)
80
Pelleting: rotor type
T-865
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
15
Pelleting-wash: duration (min)
80
Pelleting-wash: speed (g)
T-865
Commercial kit
qEV
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
<200 nm
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix/ GSTM2
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
<200
EV concentration
Yes
Particle yield
Number of particles of starting sample E08-E09
EV200034 1/1 Homo sapiens Cell culture supernatant (d)(U)C
DG
Filtration
Xiaohui Chen 2020 56%

Study summary

Full title
All authors
Xiaohui Chen, Mei Jia, Lianhua Liu, Xiaopei Qiu, Hong Zhang, Xingle Yu, Wei Gu, Guangchao Qing, Qingmei Li, Xiaolin Hu, Ruixuan Wang, Xianxian Zhao, Liangliang Zhang, Xianfeng Wang, Colm Durkan, Nan Wang, Guixue Wang, Yang Luo
Journal
Small
Abstract
Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for eluci (show more...)Direct tracing of small extracellular vesicle (sEV) cargoes holds unprecedented importance for elucidating the mechanisms involved in intercellular communication. However, high-fidelity determination of sEVs' molecular cargoes in situ has yet to be achieved due to the difficulty in transporting molecular probes into intact sEVs. Herein, a fLuorescent Intracellular-Guided Hairpin-Tetrahedron (fLIGHT) nanoprobe is described for direct visualization of sEV microRNAs in situ. Integrating the advantages of nondestructive sEV penetration via DNA origami and single-nucleotide discrimination as well as wash-free fluorescence readout using a hairpin probe, the proposed approach enables high-fidelity fluorescence visualization of sEVs' microRNA without RNA extraction or leakage, demonstrating the potential of on-site tracing of sEV cargoes. This strategy opens an avenue to establishing universal molecular detection and labeling platforms that can facilitate both sEV-derived fundamental biological studies and molecular diagnostics. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
A549
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Filtration
Protein markers
EV: TSG101/ CD9/ CD81/ CD63/ actin-beta
non-EV: None
Proteomics
no
EV density (g/ml)
1.15
Show all info
Study aim
New methodological development/Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
A549
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Not reported
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5
Highest density fraction
40
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Rotor type
Not specified
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
2
Fraction processing
Ultracentrifugation
Pelleting: duration (min)
60
Pelleting: rotor type
Not reported
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
TSG101/ CD9/ CD81/ CD63/ actin-beta
Flow cytometry
Hardware adjustments
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
104
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200024 1/2 Homo sapiens Blood plasma (d)(U)C
Otahal, Alexander 2020 56%

Study summary

Full title
All authors
Alexander Otahal, Karina Kramer, Olga Kuten-Pella, René Weiss, Christoph Stotter, Zsombor Lacza, Viktoria Weber, Stefan Nehrer and Andrea De Luna
Journal
Front. Bioeng. Biotechnol.
Abstract
Autologous blood products gain increasing interest in the field of regenerative medicine as well as (show more...)Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA. (hide)
EV-METRIC
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Alix/ CD63/ CD9
non-EV: APOA1/ APOB100
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Alix
Not detected contaminants
APOA1/ APOB100
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
160
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Gallios
Hardware adjustment
Calibration bead size
1
EV200024 2/2 Homo sapiens Serum (d)(U)C Otahal, Alexander 2020 56%

Study summary

Full title
All authors
Alexander Otahal, Karina Kramer, Olga Kuten-Pella, René Weiss, Christoph Stotter, Zsombor Lacza, Viktoria Weber, Stefan Nehrer and Andrea De Luna
Journal
Front. Bioeng. Biotechnol.
Abstract
Autologous blood products gain increasing interest in the field of regenerative medicine as well as (show more...)Autologous blood products gain increasing interest in the field of regenerative medicine as well as in orthopedics, aesthetic surgery, and cosmetics. Currently, citrate-anticoagulated platelet-rich plasma (CPRP) preparations are often applied in osteoarthritis (OA), but more physiological and cell-free alternatives such as hyperacute serum (hypACT) are under development. Besides growth factors, blood products also bring along extracellular vesicles (EVs) packed with signal molecules, which open up a new level of complexity at evaluating the functional spectrum of blood products. Large proportions of EVs originated from platelets in CPRP and hypACT, whereas very low erythrocyte and monocyte-derived EVs were detected via flow cytometry. EV treatment of chondrocytes enhanced the expression of anabolic markers type II collagen, SRY-box transcription factor 9 (SOX9), and aggrecan compared to full blood products, but also the catabolic marker and tissue remodeling factor matrix metalloproteinase 3, whereas hypACT EVs prevented type I collagen expression. CPRP blood product increased SOX9 protein expression, in contrast to hypACT blood product. However, hypACT EVs induced SOX9 protein expression while preventing interleukin-6 secretion. The results indicate that blood EVs are sufficient to induce chondrogenic gene expression changes in OA chondrocytes, while preventing proinflammatory cytokine release compared to full blood product. This highlights the potential of autologous blood-derived EVs as regulators of cartilage extracellular matrix metabolism and inflammation, as well as candidates for new cell-free therapeutic approaches for OA. (hide)
EV-METRIC
56% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: Alix/ CD63/ CD9
non-EV: APOA1/ APOB100
Proteomics
no
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
MLA-80
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD9/ CD63/ Alix
Not detected contaminants
APOA1/ APOB100
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
170
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Gallios
Hardware adjustment
Calibration bead size
1
Report type
Modus
Reported size (nm)
170
EV200016 1/2 Bos taurus Cell culture supernatant (d)(U)C
ExoQuick
Filtration
Samuel Gebremedhn 2020 56%

Study summary

Full title
All authors
Samuel Gebremedhn, Ahmed Gad, Hoda Samir Aglan, Jozef Laurincik, Radek Prochazka, Dessie Salilew-Wondim, Michael Hoelker, Karl Schellander, Dawit Tesfaye
Journal
Sci Rep
Abstract
Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, wh (show more...)Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Ovarian granulosa cells
Sample origin
Granulosa cells subjected to normal temperature (37OC)
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
ExoQuick
Filtration
Protein markers
EV: CD63
non-EV: Cytochrome C
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Bos taurus
Sample Type
Cell culture supernatant
Sample Condition
Granulosa cells subjected to normal temperature (37OC)
EV-producing cells
Ovarian granulosa cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
5
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63
Not detected contaminants
Cytochrome C
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
131
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 5.98E+08
EM
EM-type
Transmission-EM
Image type
Close-up
EV200016 2/2 Bos taurus Cell culture supernatant (d)(U)C
ExoQuick
Filtration
Samuel Gebremedhn 2020 56%

Study summary

Full title
All authors
Samuel Gebremedhn, Ahmed Gad, Hoda Samir Aglan, Jozef Laurincik, Radek Prochazka, Dessie Salilew-Wondim, Michael Hoelker, Karl Schellander, Dawit Tesfaye
Journal
Sci Rep
Abstract
Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, wh (show more...)Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Ovarian granulosa cells
Sample origin
Granulosa cells subjected to higher temperature (42OC)
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
ExoQuick
Filtration
Protein markers
EV: CD63
non-EV: Cytochrome
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Bos taurus
Sample Type
Cell culture supernatant
Sample Condition
Granulosa cells subjected to higher temperature (42OC)
EV-producing cells
Ovarian granulosa cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
5
Wash: time (min)
70
Wash: Rotor Type
SW 55 Ti
Wash: speed (g)
120000
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63
Not detected contaminants
Cytochrome
Characterization: Particle analysis
PMID previous EV particle analysis
Extra particle analysis
NTA
Report type
Modus
Reported size (nm)
129
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 7.23E+08
EM
EM-type
Transmission-EM
Image type
Close-up
EV200012 2/2 Rattus norvegicus Cell culture supernatant UF Doreen Matthies 2020 56%

Study summary

Full title
All authors
Doreen Matthies, Nathanael Y J Lee, Ian Gatera, H Amalia Pasolli, Xiaowei Zhao, Hui Liu, Deepika Walpita, Zhe Liu, Zhiheng Yu, Maria S Ioannou
Journal
Sci Rep
Abstract
Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been imp (show more...)Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Primary cortical neurons
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
Protein markers
EV: tubulin/ Flotillin1/ syntenin
non-EV: gp96
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Rattus norvegicus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Primary cortical neurons
EV-harvesting Medium
Serum free medium
Cell number specification
No
Separation Method
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Characterization: Protein analysis
PMID previous EV protein analysis
Extra characterization
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ syntenin
Not detected EV-associated proteins
tubulin
Not detected contaminants
gp96
PMID previous EV particle analysis
Extra particle analysis
EM
EM-type
Cryo-EM
Image type
Close-up
Report size (nm)
147.82+/-95.72
EV concentration
Yes
EV200000 1/4 Homo sapiens pleural effusion (d)(U)C Ping, Luo 2020 56%

Study summary

Full title
All authors
Ping Luo, Kaimin Mao, Juanjuan Xu, Feng Wu, Xuan Wang, Sufei Wang, Mei Zhou, Limin Duan, Qi Tan, Guangzhou Ma, Guanghai Yang, Ronghui Du, Hai Huang, Qi Huang, Yumei Li, Mengfei Guo, Yang Jin
Journal
J Extracell Vesicles
Abstract
Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of (show more...)Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of tuberculosis pleural effusion (TPE) and malignancy pleural effusion (MPE) remain challenging. Although extracellular vesicles (EVs) have been confirmed as promising sources of disease biomarkers, little is known about the metabolite compositions of its subpopulations and their roles in the diagnosis of pleural effusion. Here, we performed metabolomics and lipidomics analysis to investigate the metabolite characteristics of two EV subpopulations derived from pleural effusion by differential ultracentrifugation, namely large EVs (lEVs, pelleted at 20,000 × g) and small EVs (sEVs, pelleted at 110,000 × g), and assessed their metabolite differences between tuberculosis and malignancy. A total of 579 metabolites, including amino acids, acylcarnitines, organic acids, steroids, amides and various lipid species, were detected. The results showed that the metabolic profiles of lEVs and sEVs overlapped with and difference from each other but significantly differed from those of pleural effusion. Additionally, different type of vesicles and pleural effusion showed unique metabolic enrichments. Furthermore, lEVs displayed more significant and larger metabolic alterations between the tuberculosis and malignancy groups, and their differential metabolites were more closely related to clinical parameters than those of sEV. Finally, a panel of four biomarker candidates, including phenylalanine, leucine, phosphatidylcholine 35:0, and sphingomyelin 44:3, in pleural lEVs was defined based on the comprehensive discovery and validation workflow. This panel showed high performance for distinguishing TPE and MPE, particularly in patients with delayed or missed diagnosis, such as the area under the receiver-operating characteristic curve (AUC) >0.95 in both sets. We conducted comprehensive metabolic profiling analysis of EVs, and further explored the metabolic reprogramming of tuberculosis and malignancy at the level of metabolites in lEVs and sEVs, providing insight into the mechanism of pleural effusion, and identifying novel biomarkers for diagnosing TPE and MPE. (hide)
EV-METRIC
56% (57th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
pleural effusion
Sample origin
tuberculosis
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD81/ CD63/ CD9
non-EV: calnexin/ GM130
Proteomics
no
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
pleural effusion
Sample Condition
tuberculosis
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
30
Wash: time (min)
90
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
110000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
50-200 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ TSG101/ CD81
Not detected EV-associated proteins
CD9
Not detected contaminants
calnexin/ GM130
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
136.1
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 182567942
EV200000 2/4 Homo sapiens pleural effusion (d)(U)C Ping, Luo 2020 56%

Study summary

Full title
All authors
Ping Luo, Kaimin Mao, Juanjuan Xu, Feng Wu, Xuan Wang, Sufei Wang, Mei Zhou, Limin Duan, Qi Tan, Guangzhou Ma, Guanghai Yang, Ronghui Du, Hai Huang, Qi Huang, Yumei Li, Mengfei Guo, Yang Jin
Journal
J Extracell Vesicles
Abstract
Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of (show more...)Pleural effusion is a common respiratory disease worldwide; however, rapid and accurate diagnoses of tuberculosis pleural effusion (TPE) and malignancy pleural effusion (MPE) remain challenging. Although extracellular vesicles (EVs) have been confirmed as promising sources of disease biomarkers, little is known about the metabolite compositions of its subpopulations and their roles in the diagnosis of pleural effusion. Here, we performed metabolomics and lipidomics analysis to investigate the metabolite characteristics of two EV subpopulations derived from pleural effusion by differential ultracentrifugation, namely large EVs (lEVs, pelleted at 20,000 × g) and small EVs (sEVs, pelleted at 110,000 × g), and assessed their metabolite differences between tuberculosis and malignancy. A total of 579 metabolites, including amino acids, acylcarnitines, organic acids, steroids, amides and various lipid species, were detected. The results showed that the metabolic profiles of lEVs and sEVs overlapped with and difference from each other but significantly differed from those of pleural effusion. Additionally, different type of vesicles and pleural effusion showed unique metabolic enrichments. Furthermore, lEVs displayed more significant and larger metabolic alterations between the tuberculosis and malignancy groups, and their differential metabolites were more closely related to clinical parameters than those of sEV. Finally, a panel of four biomarker candidates, including phenylalanine, leucine, phosphatidylcholine 35:0, and sphingomyelin 44:3, in pleural lEVs was defined based on the comprehensive discovery and validation workflow. This panel showed high performance for distinguishing TPE and MPE, particularly in patients with delayed or missed diagnosis, such as the area under the receiver-operating characteristic curve (AUC) >0.95 in both sets. We conducted comprehensive metabolic profiling analysis of EVs, and further explored the metabolic reprogramming of tuberculosis and malignancy at the level of metabolites in lEVs and sEVs, providing insight into the mechanism of pleural effusion, and identifying novel biomarkers for diagnosing TPE and MPE. (hide)
EV-METRIC
56% (57th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
pleural effusion
Sample origin
tuberculosis
Focus vesicles
Other / small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: CD81/ TSG101/ CD63/ CD9
non-EV: calnexin/ GM130
Proteomics
no
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
pleural effusion
Sample Condition
tuberculosis
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
30
Wash: time (min)
90
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
110000
EV-subtype
Distinction between multiple subtypes
Size
Used subtypes
200-1000 nm
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Not detected EV-associated proteins
CD81
Not detected contaminants
calnexin/ GM130
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
224.2
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 244682871
EV190089 1/1 Homo sapiens Cell culture supernatant (d)(U)C Guowen Hu 2020 56%

Study summary

Full title
All authors
Guowen Hu, Yuguo Xia, Juntao Zhang, Yu Chen, Ji Yuan, Xin Niu, Bizeng Zhao, Qing Li, Yang Wang, Zhifeng Deng
Journal
Advanced Science
Abstract
Vascular dementia (VD) is one of the most common types of dementia, however, the intrinsic mechanism (show more...)Vascular dementia (VD) is one of the most common types of dementia, however, the intrinsic mechanism is unclear and there is still lack of effective medications. In this study, the VD rats exhibit a progressive cognitive impairment, as well as a time‐related increasing in hippocampal neural stem cells (H‐NSCs) senescence, lost and neurogenesis decline. Then, embryonic stem cell‐derived small extracellular vesicles (ESC‐sEVs) are intravenously injected into VD rats. ESC‐sEVs treatment significantly alleviates H‐NSCs senescence, recovers compromised proliferation and neuron differentiation capacity, and reverses cognitive impairment. By microarray analysis and RT‐qPCR it is identified that several miRNAs including miR‐17‐5p, miR‐18a‐5p, miR‐21‐5p, miR‐29a‐3p, and let‐7a‐5p, that can inhibit mTORC1 activation, exist in ESC‐sEVs. ESC‐sEVs rejuvenate H‐NSCs senescence partly by transferring these miRNAs to inhibit mTORC1 activation, promote transcription factor EB (TFEB) nuclear translocation and lysosome resumption. Taken together, these data indicate that H‐NSCs senescence cause cell depletion, neurogenesis reduction, and cognitive impairment in VD. ESC‐sEVs treatment ameliorates H‐NSCs senescence by inhibiting mTORC1 activation, and promoting TFEB nuclear translocation and lysosome resumption, thereby reversing senescence‐related neurogenesis dysfunction and cognitive impairment in VD. The application of ESC‐sEVs may be a novel cell‐free therapeutic tool for patients with VD, as well as other aging‐related diseases. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
Embryonic stem cells
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD63/ CD9
non-EV: GM130
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Embryonic stem cells
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
114
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
38.5
Wash: time (min)
114
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101
Not detected contaminants
GM130
EV190069 3/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Mariscal, Javier 2020 56%

Study summary

Full title
All authors
Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
DU145
Sample origin
DIAPH3 Knock down
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: CD9/ HSPA5
non-EV:
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
DIAPH3 Knock down
EV-producing cells
DU145
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
30
Pelleting: rotor type
SW 28
Pelleting: speed (g)
10000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
30%
Total gradient volume, incl. sample (mL)
16.2
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
SW 28
Speed (g)
100000
Duration (min)
230
Fraction volume (mL)
2.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
10000
Characterization: Protein analysis
Protein Concentration Method
Other;Pierce 660 nm
Western Blot
Detected EV-associated proteins
HSPA5
Not detected EV-associated proteins
CD9
Characterization: Particle analysis
TRPS
Report type
Modus
Reported size (nm)
1650
EV concentration
Yes
EV190069 4/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Mariscal, Javier 2020 56%

Study summary

Full title
All authors
Javier Mariscal, Tatyana Vagner, Minhyung Kim, Bo Zhou, Andrew Chin, Mandana Zandian, Michael R Freeman, Sungyong You, Andries Zijlstra, Wei Yang, Dolores Di Vizio
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer p (show more...)Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
DU145
Sample origin
DIAPH3 Knock down
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
DG
(d)(U)C
Protein markers
EV: CD9/ HSPA5
non-EV:
Proteomics
no
EV density (g/ml)
1.1
Show all info
Study aim
Biomarker/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
DIAPH3 Knock down
EV-producing cells
DU145
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
30%
Total gradient volume, incl. sample (mL)
16.2
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
SW 28
Speed (g)
100000
Duration (min)
230
Fraction volume (mL)
2.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
16
Pelleting: duration (min)
60
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Other;Pierce 660 nm
Western Blot
Detected EV-associated proteins
CD9
Not detected EV-associated proteins
HSPA5
Characterization: Particle analysis
TRPS
Report type
Modus
Reported size (nm)
116
EV concentration
Yes
EV190066 1/2 Homo sapiens Cell culture supernatant (d)(U)C
qEV
Gori, Alessandro 2020 56%

Study summary

Full title
All authors
Alessandro Gori, Alessandro Romanato, Bergamaschi Greta, Alessandro Strada, Paola Gagni, Roberto Frigerio, Dario Brambilla, Riccardo Vago, Silvia Galbiati, Silvia Picciolini, Marzia Bedoni, George G. Daaboul, Marcella Chiari, and Marina Creticha
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extrac (show more...)Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a “universal” marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
qEV
Protein markers
EV: TSG101/ Alix/ CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HEK
EV-harvesting Medium
Not specified
Separation Method
Differential ultracentrifugation
centrifugation steps
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Surespin 630 (17 ml)
Pelleting: speed (g)
28400
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ Alix
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
203
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190066 2/2 Homo sapiens Serum (d)(U)C
qEV
Gori, Alessandro 2020 56%

Study summary

Full title
All authors
Alessandro Gori, Alessandro Romanato, Bergamaschi Greta, Alessandro Strada, Paola Gagni, Roberto Frigerio, Dario Brambilla, Riccardo Vago, Silvia Galbiati, Silvia Picciolini, Marzia Bedoni, George G. Daaboul, Marcella Chiari, and Marina Creticha
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extrac (show more...)Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a “universal” marker, alternative or complementary to traditional, characteristic, surface-associated proteins. Here, we introduce the use of membrane-sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label-free, single-particle counting and sizing, and by fluorescence co-localisation immune staining with fluorescent anti-CD9/anti-CD63/anti-CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti-tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis. (hide)
EV-METRIC
56% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
qEV
Protein markers
EV: TSG101/ Alix/ CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
New methodological development
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Equal to or above 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
41900
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Alix/ CD9/ CD63/ TSG101
Not detected EV-associated proteins
TSG101/ CD63/ CD9/ Alix
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
208
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Wide-field
EV190064 2/10 Homo sapiens Urine (d)(U)C
Filtration
Dhondt B 2020 56%

Study summary

Full title
All authors
Dhondt B, Geeurickx E, Tulkens J, Van Deun J, Vergauwen G, Lippens L, Miinalainen I, Rappu P, Heino J, Ost P, Lumen N, De Wever O, Hendrix A.
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular (show more...)Extracellular vesicles (EV) are increasingly being recognized as important vehicles of intercellular communication and promising diagnostic and prognostic biomarkers in cancer. Despite this enormous clinical potential, the plethora of methods to separate EV from biofluids, providing material of highly variable purity, and lacking knowledge regarding methodological repeatability pose a barrier to clinical translation. Urine is considered an ideal proximal fluid for the study of EV in urological cancers due to its direct contact with the urogenital system. We demonstrate that density-based fractionation of urine by bottom-up Optiprep density gradient centrifugation separates EV and soluble proteins with high specificity and repeatability. Mass spectrometry-based proteomic analysis of urinary EV (uEV) in men with benign and malignant prostate disease allowed us to significantly expand the known human uEV proteome with high specificity and identifies a unique biological profile in prostate cancer not uncovered by the analysis of soluble proteins. In addition, profiling the proteome of EV separated from prostate tumour conditioned medium and matched uEV confirms the specificity of the identified uEV proteome for prostate cancer. Finally, a comparative proteomic analysis with uEV from patients with bladder and renal cancer provided additional evidence of the selective enrichment of protein signatures in uEV reflecting their respective cancer tissues of origin. In conclusion, this study identifies hundreds of previously undetected proteins in uEV of prostate cancer patients and provides a powerful toolbox to map uEV content and contaminants ultimately allowing biomarker discovery in urological cancers. (hide)
EV-METRIC
56% (90th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Urine
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: Alix/ Flotillin1/ CD9
non-EV: Tamm-Horsfall protein
Proteomics
no
Show all info
Study aim
Function/New methodological development/Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Urine
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
16
Wash: time (min)
70
Wash: Rotor Type
SW 32.1 Ti
Wash: speed (g)
110000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ CD9
Detected contaminants
Tamm-Horsfall protein
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
226.2
EV concentration
Yes
EV190060 1/4 Homo sapiens Blood plasma (d)(U)C Mari Palviainen 2020 56%

Study summary

Full title
All authors
Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide)
EV-METRIC
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD61/ CD41/ phosphatidylserine/ CD235a/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
20
Wash: time (min)
90
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD9/ CD41/ TSG101
Flow cytometry aspecific beads
Detected EV-associated proteins
CD61/ CD235a/ phosphatidylserine
Proteomics
Proteomics database
Yes:
Other 1
Flow cytometry (after non-specific association of vesicles to beads)
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
107-145
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Apogee A50
Hardware adjustment
calibration done with apogee Mix beads 80-1300 nm
Calibration bead size
80
EM
EM-type
Transmission-EM
Image type
Close-up
EV190060 2/4 Homo sapiens Blood plasma (d)(U)C Mari Palviainen 2020 56%

Study summary

Full title
All authors
Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide)
EV-METRIC
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD61/ CD41/ phosphatidylserine/ CD235a/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
20
Wash: time (min)
90
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD9/ CD41/ TSG101
Flow cytometry aspecific beads
Detected EV-associated proteins
CD61/ CD235a/ phosphatidylserine
Proteomics
Proteomics database
Yes:
Other 1
Flow cytometry (after non-specific association of vesicles to beads)
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
117-162
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
apogee A50
Hardware adjustment
calibration done with apogee Mix beads 80-1300 nm
Calibration bead size
80
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up
EV190060 3/4 Homo sapiens Blood plasma (d)(U)C Mari Palviainen 2020 56%

Study summary

Full title
All authors
Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide)
EV-METRIC
56% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD61/ CD41/ phosphatidylserine/ Cd235a/ CD9
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
20
Wash: time (min)
90
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD9/ TSG101/ CD41
Flow cytometry aspecific beads
Detected EV-associated proteins
CD61/ Cd235a/ phosphatidylserine
Proteomics
Proteomics database
Yes:
Other 1
Flow cytometry (after non-specific association of vesicles to beads)
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
106-160
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Apogee A50
Hardware adjustment
calibration done with apogee Mix beads 80-1300 nm
Calibration bead size
80
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up
EV190060 4/4 Homo sapiens Serum (d)(U)C Mari Palviainen 2020 56%

Study summary

Full title
All authors
Mari Palviainen, Mayank Saraswat, Zoltán Varga, Diána Kitka, Maarit Neuvonen, Maija Puhka, Sakari Joenväärä, Risto Renkonen, Rienk Nieuwland, Maarit Takatalo, Pia R M Siljander
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent an (show more...)Extracellular vesicles (EVs) in human blood are a potential source of biomarkers. To which extent anticoagulation affects their concentration, cellular origin and protein composition is largely unexplored. To study this, blood from 23 healthy subjects was collected in acid citrate dextrose (ACD), citrate or EDTA, or without anticoagulation to obtain serum. EVs were isolated by ultracentrifugation or by size-exclusion chromatography (SEC) for fluorescence-SEC. EVs were analyzed by micro flow cytometry, NTA, TEM, Western blot, and protein mass spectrometry. The plasma EV concentration was unaffected by anticoagulants, but serum contained more platelet EVs. The protein composition of plasma EVs differed between anticoagulants, and between plasma and serum. Comparison to other studies further revealed that the shared EV protein composition resembles the "protein corona" of synthetic nanoparticles incubated in plasma or serum. In conclusion, we have validated a higher concentration of platelet EVs in serum than plasma by contemporary EV methods. Anticoagulation should be carefully described (i) to enable study comparison, (ii) to utilize available sample cohorts, and (iii) when preparing/selecting biobank samples. Further, the similarity of the EV protein corona and that of nanoparticles implicates that EVs carry both intravesicular and extravesicular cargo, which will expand their applicability for biomarker discovery. (hide)
EV-METRIC
56% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Protein markers
EV: TSG101/ CD235a/ CD41/ CD9/ phosphatidylserine
non-EV:
Proteomics
yes
Show all info
Study aim
Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
110000
Wash: volume per pellet (ml)
20
Wash: time (min)
90
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
110000
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Detected EV-associated proteins
CD41/ CD9/ TSG101
Flow cytometry aspecific beads
Detected EV-associated proteins
CD41/ CD235a/ phosphatidylserine
Proteomics
Proteomics database
Yes:
Other 1
Flow cytometry (after non-specific association of vesicles to beads)
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
88-128
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
Apogee A50
Hardware adjustment
calibration done with apogee Mix beads 80-1300 nm
Calibration bead size
80
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up
EV190059 1/1 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Gong, Liangzhi 2020 56%

Study summary

Full title
All authors
Liangzhi Gong, Bi Chen, Juntao Zhang, Yongjin Sun, Ji Yuan, Xin Niu, Guowen Hu, Yu Chen, Zongping Xie, Zhifeng Deng, Qing Li, Yang Wang
Journal
J Extracell Vesicles
Abstract
Tissue-resident stem cell senescence leads to stem cell exhaustion, which is a major cause of physio (show more...)Tissue-resident stem cell senescence leads to stem cell exhaustion, which is a major cause of physiological and pathological ageing. Stem cell-derived extracellular vesicles (SC-EVs) have been reported in preclinical studies to possess therapeutic potential for diverse diseases. However, whether SC-EVs can rejuvenate senescent tissue stem cells to prevent age-related disorders still remains unknown. Here, we show that chronic application of human embryonic stem cell-derived small extracellular vesicles (hESC-sEVs) rescues the function of senescent bone marrow mesenchymal stem cells (BM-MSCs) and prevents age-related bone loss in ageing mice. Transcriptome analysis revealed that hESC-sEVs treatment upregulated the expression of genes involved in antiaging, stem cell proliferation and osteogenic differentiation in BM-MSCs. Furthermore, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis identified 4122 proteins encapsulated in hESC-sEVs. Bioinformatics analysis predicted that the protein components in the hESCs-sEVs function in a synergistic way to induce the activation of several canonical signalling pathways, including Wnt, Sirtuin, AMPK, PTEN signalling, which results in the upregulation of antiaging genes in BM-MSCs and then the recovery of senescent BM-MSCs function. Collectively, our findings reveal the effect of hESC-sEVs in reversing BM-MSCs senescence and age-related osteogenic dysfunction, thereby preventing age-related bone loss. Because hESC-sEVs could alleviate senescence of tissue-resident stem cells, they might be promising therapeutic candidates for age-related diseases. (hide)
EV-METRIC
56% (84th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
H9
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
Filtration
Protein markers
EV: TSG101/ Alix/ CD63/ CD9
non-EV: GM130
Proteomics
yes
Show all info
Study aim
Function
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
H9
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
114
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
38
Wash: time (min)
114
Wash: Rotor Type
SW 32 Ti
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm0.2µm > x > 0.1µm
EV-subtype
Distinction between multiple subtypes
Used subtypes
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ Alix
Not detected contaminants
GM130
Proteomics
Proteomics database
No
Other particle analysis name(1)
qNano
Report type
Size range/distribution
Report size
75-200
EV-concentration
Yes
EV200117 2/6 Homo sapiens Cell culture supernatant (d)(U)C
UF
qEV
Cocozza, Federico 2020 50%

Study summary

Full title
All authors
Federico Cocozza, Nathalie Névo, Ester Piovesana, Xavier Lahaye, Julian Buchrieser, Olivier Schwartz, Nicolas Manel, Mercedes Tkach, Clotilde Théry, Lorena Martin‐Jaular
Journal
J Extracell Vesicles
Abstract
SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 an (show more...)SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by host TMPRSS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2‐EVs are efficient decoys for SARS‐CoV‐2 S protein‐containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is much more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK
Sample origin
HEK MOCK
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Ultrafiltration
qEV
Protein markers
EV: CD81/ ACE2/ ADAM10/ CD63/ syntenin-1/ HSP70
non-EV: AChe
Proteomics
no
Show all info
Study aim
Function/New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
HEK MOCK
EV-producing cells
HEK
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD63/ syntenin-1/ ACE2/ ADAM10/ CD81/ HSP70
Not detected contaminants
AChe
ELISA
Detected EV-associated proteins
ACE2
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
CD63/ CD81/ syntenin-1
Detected EV-associated proteins
CD81/ CD63/ syntenin-1
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
141.8
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 5.00E+08
EV200117 3/6 Homo sapiens Cell culture supernatant (d)(U)C
UF
qEV
Cocozza, Federico 2020 50%

Study summary

Full title
All authors
Federico Cocozza, Nathalie Névo, Ester Piovesana, Xavier Lahaye, Julian Buchrieser, Olivier Schwartz, Nicolas Manel, Mercedes Tkach, Clotilde Théry, Lorena Martin‐Jaular
Journal
J Extracell Vesicles
Abstract
SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 an (show more...)SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by host TMPRSS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2‐EVs are efficient decoys for SARS‐CoV‐2 S protein‐containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is much more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK
Sample origin
HEK ACE2
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Ultrafiltration
qEV
Protein markers
EV: CD81/ ACE2/ ADAM10/ CD63/ syntenin-1/ HSP70
non-EV: AChe
Proteomics
no
Show all info
Study aim
Function/New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
HEK ACE2
EV-producing cells
HEK
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD63/ syntenin-1/ ACE2/ ADAM10/ CD81/ HSP70
Not detected contaminants
AChe
ELISA
Detected EV-associated proteins
ACE2
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
CD63/ CD81/ syntenin-1
Detected EV-associated proteins
CD81/ CD63/ syntenin-1
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
145.5
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 7.00E+08
EV200117 4/6 Homo sapiens Cell culture supernatant (d)(U)C
UF
qEV
Cocozza, Federico 2020 50%

Study summary

Full title
All authors
Federico Cocozza, Nathalie Névo, Ester Piovesana, Xavier Lahaye, Julian Buchrieser, Olivier Schwartz, Nicolas Manel, Mercedes Tkach, Clotilde Théry, Lorena Martin‐Jaular
Journal
J Extracell Vesicles
Abstract
SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 an (show more...)SARS‐CoV‐2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by host TMPRSS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2‐EVs are efficient decoys for SARS‐CoV‐2 S protein‐containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is much more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HEK
Sample origin
HEK ACE2 and TMPRSS2
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Ultrafiltration
qEV
Protein markers
EV: CD81/ ACE2/ ADAM10/ CD63/ syntenin-1/ HSP70
non-EV: AChe
Proteomics
no
Show all info
Study aim
Function/New methodological development
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
HEK ACE2 and TMPRSS2
EV-producing cells
HEK
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Ultra filtration
Cut-off size (kDa)
10
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
syntenin-1/ ACE2/ ADAM10/ CD63/ CD81/ HSP70
Not detected contaminants
AChe
ELISA
Detected EV-associated proteins
ACE2
Flow cytometry
Hardware adjustments
Detected EV-associated proteins
CD63/ CD81/ syntenin-1
Detected EV-associated proteins
CD81/ CD63/ syntenin-1
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
153.9
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 6.00E+08
EV200068 1/5 Homo sapiens Blood plasma (d)(U)C
SEC (non-commercial)
Filtration
Linda Hofmann 2020 50%

Study summary

Full title
All authors
Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki
Journal
Int J Mol Sci
Abstract
Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
SEC (non-commercial)
Filtration
Protein markers
EV: TSG101/ CD81/ CD63/ CD9/ CD16
non-EV: Grp94/ ApoA1
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ CD81
Not detected contaminants
ApoA1/ Grp94
Flow cytometry aspecific beads
Detected EV-associated proteins
CD16
Flow cytometry specific beads
Selected surface protein(s)
CD44v3
Detected EV-associated proteins
CD16
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-150
EM
EM-type
Transmission-EM
Image type
Wide-field
EV200068 2/5 Homo sapiens Blood plasma (d)(U)C
SEC (non-commercial)
Filtration
Linda Hofmann 2020 50%

Study summary

Full title
All authors
Linda Hofmann, Sonja Ludwig, Patrick J Schuler, Thomas K Hoffmann, Cornelia Brunner, Marie-Nicole Theodoraki
Journal
Int J Mol Sci
Abstract
Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignan (show more...)Head and neck squamous cell carcinomas (HNSCC) are highly immune suppressive and aggressive malignancies. As part of the tumor microenvironment, exosomes contribute to this immune suppression. The Fc receptor CD16 is widely expressed on monocytes, neutrophils, and natural killer (NK) cells and is involved in antibody-dependent cell-mediated cytotoxicity (ADCC). Here, surface levels of CD16 on total exosomes and tumor-derived exosomes (TEX) from plasma of HNSCC patients were analyzed regarding their potential as liquid biomarkers for disease stage. Exosomes were isolated from plasma using mini size exclusion chromatography. TEX were enriched by immune affinity capture with CD44v3 antibodies. On-bead flow cytometry was used to measure CD16 levels on total exosomes and TEX. The results were correlated with clinicopathological parameters. Total exosomes from HNSCC patients had significantly higher CD16 levels compared to TEX. Further, CD16 surface levels of total exosomes, but not TEX, correlated with clinicopathological parameters. Patients with advanced tumor stages T3/4 and Union for International Cancer Control (UICC) stages III/IV had significantly higher CD16 levels on total exosomes compared to patients with early tumor stages T1/2 and UICC stages I/II, respectively. Overall, CD16 positive exosomes have the potential as liquid biomarkers for HNSCC tumor stage and aggressiveness. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
HNSCC
Focus vesicles
exosome
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
SEC (non-commercial)
Filtration
Protein markers
EV: TSG101/ CD81/ CD63/ CD9/ CD16
non-EV: Grp94/ ApoA1
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
HNSCC
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Resin type
Sepharose CL-2B
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ TSG101/ CD81
Not detected contaminants
ApoA1/ Grp94
Flow cytometry aspecific beads
Detected EV-associated proteins
CD16
Flow cytometry specific beads
Selected surface protein(s)
CD44v3
Detected EV-associated proteins
CD16
EV200063 1/2 Homo sapiens Blood plasma qEV Silvia Picciolini 2020 50%

Study summary

Full title
All authors
Silvia Picciolini,Alice Gualerzi,Cristiano Carlomagno,Monia Cabinio,Stefano Sorrentino,Francesca Baglio,Marzia Bedoni
Journal
ACS Nano
Abstract
One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible (show more...)One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible and sensitive biomarkers for the diagnosis, the prediction of the disease progression rate and the evaluation of rehabilitative and pharmacological treatments. Extracellular Vesicles (EVs) are nanoscales particles released by body cells studied as promising biomarkers of AD as they are involved in the onset and progression of the disease. In the strive for a reliable and sensitive method to analyze EVs, we applied our recently developed biosensor based on Surface Plasmon Resonance imaging (SPRi) technology for the identification and profiling of neural EVs populations circulating in the plasma of 10 AD patients and 10 healthy subjects. The SPRi-array was designed to separate simultaneously EVs released by neurons, astrocytes, microglia and oligodendrocytes, and to evaluate the presence and the relative amount of specific surface molecules related to pathological processes including translocator protein (TSPO), β-Amyloid and ganglioside M1. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
qEV
Protein markers
EV: CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB
non-EV: Ig
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Detected EV-associated proteins
CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB
Not detected contaminants
Ig
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
166,45
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV200063 2/2 Homo sapiens Blood plasma qEV Silvia Picciolini 2020 50%

Study summary

Full title
All authors
Silvia Picciolini,Alice Gualerzi,Cristiano Carlomagno,Monia Cabinio,Stefano Sorrentino,Francesca Baglio,Marzia Bedoni
Journal
ACS Nano
Abstract
One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible (show more...)One of the main hurdles in the study of Alzheimer’s Disease (AD) is the lack of easily accessible and sensitive biomarkers for the diagnosis, the prediction of the disease progression rate and the evaluation of rehabilitative and pharmacological treatments. Extracellular Vesicles (EVs) are nanoscales particles released by body cells studied as promising biomarkers of AD as they are involved in the onset and progression of the disease. In the strive for a reliable and sensitive method to analyze EVs, we applied our recently developed biosensor based on Surface Plasmon Resonance imaging (SPRi) technology for the identification and profiling of neural EVs populations circulating in the plasma of 10 AD patients and 10 healthy subjects. The SPRi-array was designed to separate simultaneously EVs released by neurons, astrocytes, microglia and oligodendrocytes, and to evaluate the presence and the relative amount of specific surface molecules related to pathological processes including translocator protein (TSPO), β-Amyloid and ganglioside M1. (hide)
EV-METRIC
50% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Blood plasma
Sample origin
Alzheimer disease
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
qEV
Protein markers
EV: CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB
non-EV: Ig
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Alzheimer disease
Separation Method
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
BCA
Detected EV-associated proteins
CD9/ CD171/ Glast/ PLP1/ CD11b/ EphrinB
Not detected contaminants
Ig
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
183,28
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
EV190100 1/10 Homo sapiens Cell culture supernatant ExoQuick Chaoliang Liao 2020 50%

Study summary

Full title
All authors
Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang
Journal
J Pharm Sci
Abstract
Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
CNE1
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
ExoQuick
Protein markers
EV: HSP70/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
CNE1
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
21.04-255.6
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
80-100
EV190100 2/10 Homo sapiens Cell culture supernatant ExoQuick Chaoliang Liao 2020 50%

Study summary

Full title
All authors
Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang
Journal
J Pharm Sci
Abstract
Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
CNE1-LMP1
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
ExoQuick
Protein markers
EV: HSP70/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
CNE1-LMP1
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Not detected contaminants
Calnexin
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
80-100
EV190100 6/10 Homo sapiens Cell culture supernatant ExoQuick Chaoliang Liao 2020 50%

Study summary

Full title
All authors
Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang
Journal
J Pharm Sci
Abstract
Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
HKI
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
ExoQuick
Protein markers
EV: HSP70/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HKI
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
24.3-199.2
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
80-100
EV190100 8/10 Homo sapiens Cell culture supernatant ExoQuick Chaoliang Liao 2020 50%

Study summary

Full title
All authors
Chaoliang Liao, Qin Zhou, Zhibao Zhang, Xia Wu, Zhuan Zhou, Bo Li, Jinwu Peng, Liangfang Shen, Dan Li, Xiangjian Luo, Lifang Yang
Journal
J Pharm Sci
Abstract
Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cel (show more...)Increasing evidence indicates that extracellular vesicles (EVs) play an important role in cancer cell-to-cell communication. The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1), which is closely associated with nasopharyngeal carcinoma (NPC) pathogenesis, can trigger multiple cell signaling pathways that affect cell progression. Several reports have shown that LMP1 promotes EV secretion, and LMP1 trafficking by EVs can enhances cancer progression and metastasis. However, the molecular mechanism by which LMP1 promotes EV secretion is not well understood. In the present study, we found that LMP1 promotes EV secretion by upregulated syndecan-2 (SDC2) and synaptotagmin-like-4 (SYTL4) through nuclear factor (NF)-κB signaling in NPC cells. Further study indicated that SDC2 interacted with syntenin, which promoted the formation of the EVs, and SYTL4 is associated with the release of EVs. Moreover, we found that stimulation of EV secretion by LMP1 can enhance the proliferation and invasion ability of recipient NPC cells and tumor growth in vivo. In summary, we found a new mechanism by which LMP1 upregulates SDC2 and SYTL4 through NF-κB signaling to promote EV secretion, and further enhance cancer progression of NPC. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
C666-1
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
ExoQuick
Protein markers
EV: HSP70/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function/Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
C666-1
EV-harvesting Medium
Serum-containing, but physical separation of serum EVs and secreted EVs (e.g. Bioreactor flask)
Separation Method
Commercial kit
ExoQuick
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ HSP70
Not detected contaminants
Calnexin
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
13.54-225.9
EV concentration
Yes
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
80-100
EV190099 1/3 Mus musculus Serum Filtration
qEV
Anastasi, Federica 2020 50%

Study summary

Full title
All authors
Federica Anastasi, Francesco Greco, Marialaura Dilillo, Eleonora Vannini, Valentina Cappello, Laura Baroncelli, Mario Costa, Mauro Gemmi, Matteo Caleo & Liam A. McDonnell
Journal
Sci Rep
Abstract
Longitudinal analysis of disease models enables the molecular changes due to disease progression or (show more...)Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV’s) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV’s both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV’s isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1). (hide)
EV-METRIC
50% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Filtration
qEV
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
New methodological development/Biomarker/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Filtration steps
0.22µm or 0.2µm
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Proteomics
Proteomics database
No
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
45
EV190099 2/3 Mus musculus Serum Filtration
Total Exosome Isolation
Anastasi, Federica 2020 50%

Study summary

Full title
All authors
Federica Anastasi, Francesco Greco, Marialaura Dilillo, Eleonora Vannini, Valentina Cappello, Laura Baroncelli, Mario Costa, Mauro Gemmi, Matteo Caleo & Liam A. McDonnell
Journal
Sci Rep
Abstract
Longitudinal analysis of disease models enables the molecular changes due to disease progression or (show more...)Longitudinal analysis of disease models enables the molecular changes due to disease progression or therapeutic intervention to be better resolved. Approximately 75 µl of serum can be drawn from a mouse every 14 days. To date no methods have been reported that are able to analyze the proteome of small extracellular vesicles (sEV’s) from such low serum volumes. Here we report a method for the proteomics analysis of sEV's from 50 µl of serum. Two sEV isolation procedures were first compared; precipitation based purification (PPT) and size exclusion chromatography (SEC). The methodological comparison confirmed that SEC led to purer sEV’s both in terms of size and identified proteins. The procedure was then scaled down and the proteolytic digestion further optimized. The method was then applied to a longitudinal study of serum-sEV proteome changes in a glioblastoma multiforme (GBM) mouse model. Serum was collected at multiple time points, sEV’s isolated and their proteins analyzed. The protocol enabled 274 protein groups to be identified and quantified. The longitudinal analysis revealed 25 deregulated proteins in GBM serum sEV's including proteins previously shown to be associated with GBM progression and metastasis (Myh9, Tln-1, Angpt1, Thbs1). (hide)
EV-METRIC
50% (91st percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Serum
Sample origin
Control condition
Focus vesicles
small extracellular vesicles
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Filtration
Total Exosome Isolation
Protein markers
EV:
non-EV:
Proteomics
yes
Show all info
Study aim
New methodological development/Biomarker/Identification of content (omics approaches)
Sample
Species
Mus musculus
Sample Type
Serum
Sample Condition
Control condition
Separation Method
Filtration steps
0.22µm or 0.2µm
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
microBCA
Proteomics
Proteomics database
No
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
37
EV190091 1/1 Homo sapiens Cell culture supernatant (d)(U)C
SEC
Getnet Midekessa 2020 50%

Study summary

Full title
All authors
Getnet Midekessa, Kasun Godakumara, James Ord, Janeli Viil, Freddy Lättekivi, Keerthie Dissanayake, Sergei Kopanchuk, Ago Rinken, Aneta Andronowska, Sourav Bhattacharjee, Toonika Rinken, Alireza Fazeli
Journal
ACS Omega
Abstract
Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in i (show more...)Extracellular vesicles (EVs), including exosomes and microvesicles (<200 nm), play a vital role in intercellular communication and carry a net negative surface charge under physiological conditions. Zeta potential (ZP) is a popular method to measure the surface potential of EVs, while used as an indicator of surface charge, and colloidal stability influenced by surface chemistry, bioconjugation, and the theoretical model applied. Here, we investigated the effects of such factors on ZP of well-characterized EVs derived from the human choriocarcinoma JAr cells. The EVs were suspended in phosphate-buffered saline (PBS) of various phosphate ionic concentrations (0.01, 0.1, and 1 mM), with or without detergent (Tween-20), or in the presence (10 mM) of different salts (NaCl, KCl, CaCl2, and AlCl3) and at different pH values (4, 7, and 10) while the ZP was measured. The ZP changed inversely with the buffer concentration, while Tween-20 caused a significant (p < 0.05) lowering of the ZP. Moreover, the ZP was significantly (p < 0.05) less negative in the presence of ions with higher valency (Al3+/Ca2+) than in the presence of monovalent ones (Na+/K+). Besides, the ZP of EVs became less negative at acidic pH, and vice versa. The integrated data underpins the crucial role of physicochemical attributes that influence the colloidal stability of EVs. (hide)
EV-METRIC
50% (80th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
JAR ATCC HTB-144
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(d)(U)C
SEC
Protein markers
EV: CD81/ HSP70/ CD63/ CD9
non-EV:
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
JAR ATCC HTB-144
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Not specified
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Size-exclusion chromatography
Total column volume (mL)
10.5
Sample volume/column (mL)
0.5
Resin type
Sepharose 4B
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Detected EV-associated proteins
CD9/ CD63/ HSP70/ CD81
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
15 - 500
EV concentration
Yes
EM
EM-type
Transmission-EM/ Scanning-EM
Image type
Wide-field
Report size (nm)
120 -200
EV190075 1/3 Homo sapiens Cell culture supernatant Filtration
Total Exosome Isolation
UF
Thomas E. Whittaker 2020 50%