Search > Results

You searched for: EV190002 (EV-TRACK ID)

Showing 1 - 6 of 6

Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV190002 1/6 Homo sapiens Cell culture supernatant Density gradient
Filtration
(Differential) (ultra)centrifugation
Sachiko Matsumura 2018 78%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + Filtration + (Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin
non-EV: Histon H2B
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MIA PaCa-2
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
< 1.06 g/ml
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD63/ MFGE8/ beta-actin/ HSP70/ CD81
Not detected EV-associated proteins
TSG101/ Alix
Not detected contaminants
Histon H2B
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
136
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
height: 39.1, diameter: 135.9
EV190002 2/6 Homo sapiens Cell culture supernatant Density gradient
Filtration
(Differential) (ultra)centrifugation
Sachiko Matsumura 2018 78%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + Filtration + (Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin
non-EV: Histon H2B
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MIA PaCa-2
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
ca. 1.1 g/ml
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ CD63/ MFGE8/ beta-actin/ TSG101/ HSP70/ CD81
Not detected contaminants
Histon H2B
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
124
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
height: 30.1, diameter: 96.4
EV190002 3/6 Homo sapiens Cell culture supernatant Density gradient
Filtration
(Differential) (ultra)centrifugation
Sachiko Matsumura 2018 78%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + Filtration + (Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ Src/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin/ CD9
non-EV: Argonaute2/ Histon H2B
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MIA PaCa-2
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
< 1.06 g/ml
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
CD9/ CD63/ HSP70/ MFGE8/ Src/ beta-actin/ CD81
Not detected EV-associated proteins
TSG101/ Alix
Not detected contaminants
Histon H2B/ Argonaute2
Detected EV-associated proteins
CD63
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
148
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
height: 50.5, length: 190.0
EV190002 4/6 Homo sapiens Cell culture supernatant Density gradient
Filtration
(Differential) (ultra)centrifugation
Sachiko Matsumura 2018 78%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
78% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + Filtration + (Differential) (ultra)centrifugation
Protein markers
EV: TSG101/ Src/ CD63/ MFGE8/ CD81/ Alix/ HSP70/ beta-actin/ CD9
non-EV: Argonaute2/ Histon H2B
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
MIA PaCa-2
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Equal to or above 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
ca. 1.1 g/ml
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Alix/ MFGE8/ Src/ beta-actin/ CD9/ CD63/ TSG101/ HSP70/ CD81
Not detected contaminants
Histon H2B/ Argonaute2
Detected EV-associated proteins
CD9/ CD63
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
127
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
height 32.1, length: 120.0
EV190002 5/6 Homo sapiens Cell culture supernatant Density gradient
Filtration
(Differential) (ultra)centrifugation
Sachiko Matsumura 2018 67%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
67% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Density gradient + Filtration + (Differential) (ultra)centrifugation
Protein markers
EV: CD81/ EpCAM/ CD63/ CD9
non-EV:
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HT-29
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
< 1.06 g/ml
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
EpCAM/ CD9
Not detected EV-associated proteins
CD81/ CD63
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
162
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
EV190002 6/6 Homo sapiens Cell culture supernatant (Differential) (ultra)centrifugation
Filtration
Density gradient
Sachiko Matsumura 2018 67%

Study summary

Full title
All authors
Sachiko Matsumura, Tamiko Minamisawa, Kanako Suga, Hiromi Kishita, Takanori Akagi, Takanori Ichiki, Yuki Ichikawa & Kiyotaka Shiba
Journal
J Extracell Vesicles
Abstract
Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially locate (show more...)Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes – or, more properly, small extracellular vesicles (sEVs) – which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics. However, the sEV fractions prepared from various methods are composed of different subtypes of vesicles, and knowledge about the subtypes enriched with exposed PS is still limited. Here, we differentiated sEVs from cancer cell lines by density gradient centrifugation and characterized the separated fractions by using gold-labelling of PS in atomic force microscopy, thrombin generation assay, size and zeta potential measurements, and western blot analysis. These analyses revealed a previously unreported PS+-enriched sEV subtype, which is characterized by a lower density than that of canonical exosomes (1.06 g/ml vs. 1.08 g/ml), larger size (122 nm vs. 105 nm), more negative zeta potential (−28 mV vs. −21 mV), and lower abundance of canonical exosomal markers. The identification of the PS-exposed subtype of sEVs will provide deeper insight into the role of EVs in tumour biology and enhance the development of EV-based tumour diagnosis and therapy. (hide)
EV-METRIC
67% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
Other / small extracellular vesicles
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
(Differential) (ultra)centrifugation + Filtration + Density gradient
Protein markers
EV: CD81/ EpCAM/ CD63/ CD9
non-EV:
Proteomics
no
EV density (g/ml)
1.05-1.1
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
HT-29
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Equal to or above 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Density gradient
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
8%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
32
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
1020
Fraction volume (mL)
3.2
Fraction processing
Centrifugation
Pelleting: volume per fraction
33.2
Pelleting: duration (min)
120
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
160000
Filtration steps
0.22µm or 0.2µm
EV-subtype
Distinction between multiple subtypes
Density
Used subtypes
ca. 1.1 g/ml
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD9/ CD63/ EpCAM/ CD81
Characterization: Lipid analysis
Yes
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
142
EV concentration
Yes
EM
EM-type
Atomic force-EM
Image type
Wide-field
1 - 6 of 6
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV190002
species
Homo
sapiens
sample type
Cell
culture
cell type
MIA
PaCa-2
MIA
PaCa-2
MIA
PaCa-2
MIA
PaCa-2
HT-29
HT-29
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
isolation protocol
Density
gradient
Filtration
dUC
Density
gradient
Filtration
dUC
Density
gradient
Filtration
dUC
Density
gradient
Filtration
dUC
Density
gradient
Filtration
dUC
dUC
Filtration
Density
gradient
EV subtype
<
1.06
g/ml
ca.
1.1
g/ml
<
1.06
g/ml
ca.
1.1
g/ml
<
1.06
g/ml
ca.
1.1
g/ml
Exp. nr.
1
2
3
4
5
6
EV-METRIC %
78
78
78
78
67
67