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You searched for: EV190006 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV190006 1/2 Homo sapiens Cell culture supernatant DG
(d)(U)C
Altei, Wanessa F. 2020 100%

Study summary

Full title
All authors
Wanessa F Altei, Bianca C Pachane, Patty K Dos Santos, Lígia N M Ribeiro, Bong Hwan Sung, Alissa M Weaver, Heloisa S Selistre-de-Araújo
Journal
Cell Commun Signal
Abstract
Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from (show more...)Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion: In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs. (hide)
EV-METRIC
100% (99th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
MDAMB231
Sample origin
immortalized
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: Flotillin1/ CD63/ Alix/ integrin-alpha5/ integrin-alpha2/ integrin-alphaV/ integrin-beta1/ integrin-beta3/ FN1/ COL1
non-EV: Calnexin
Proteomics
no
EV density (g/ml)
1.11
Show all info
Study aim
Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
immortalized
EV-producing cells
MDAMB231
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)
4
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Density gradient
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
Type 40
Speed (g)
100000
Duration (min)
18
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
1
Pelleting: duration (min)
180
Pelleting: rotor type
TLA-100.3
Pelleting: speed (g)
100000
Pelleting-wash: volume per pellet (mL)
3
Pelleting-wash: duration (min)
180
Pelleting-wash: speed (g)
TLA-100.3
Density cushion
Density medium
EV-subtype
Distinction between multiple subtypes
Used subtypes
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ Alix/ integrin-alpha5/ integrin-alpha2/ integrin-alphaV/ integrin-beta1/ integrin-beta3/ FN1/ COL1
Not detected EV-associated proteins
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
107
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 5.50E+08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
EV190006 2/2 Homo sapiens Cell culture supernatant (d)(U)C Altei, Wanessa F. 2020 78%

Study summary

Full title
All authors
Wanessa F Altei, Bianca C Pachane, Patty K Dos Santos, Lígia N M Ribeiro, Bong Hwan Sung, Alissa M Weaver, Heloisa S Selistre-de-Araújo
Journal
Cell Commun Signal
Abstract
Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from (show more...)Background: Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods: To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results: We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion: In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs. (hide)
EV-METRIC
78% (96th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Cell Name
MDAMB231
Sample origin
immortalized
Focus vesicles
(shedding) microvesicle
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
(Differential) (ultra)centrifugation
Protein markers
EV: Flotillin1/ CD63/ Alix/ integrin-alpha5/ integrin-alpha2/ integrin-alphaV/ integrin-beta1/ integrin-beta3/ FN1/ COL1
non-EV: Calnexin
Proteomics
no
EV density (g/ml)
Show all info
Study aim
Biogenesis/cargo sorting/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
immortalized
EV-producing cells
MDAMB231
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)
4
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
10000
Density gradient
Only used for validation of main results
Density medium
Type
Number of initial discontinuous layers
Lowest density fraction
Highest density fraction
Total gradient volume, incl. sample (mL)
Sample volume (mL)
Orientation
Rotor type
Speed (g)
Duration (min)
Fraction volume (mL)
Fraction processing
Pelleting: volume per fraction
Pelleting: duration (min)
Pelleting: rotor type
Pelleting: speed (g)
Pelleting-wash: volume per pellet (mL)
Pelleting-wash: duration (min)
Pelleting-wash: speed (g)
Density cushion
Density medium
EV-subtype
Distinction between multiple subtypes
Used subtypes
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ integrin-alpha5/ integrin-beta1
Not detected EV-associated proteins
CD63/ Alix/ FN1/ COL1
Not detected contaminants
Calnexin
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Median
Reported size (nm)
206
EV concentration
Yes
Particle yield
Yes, as number of particles per million cells 9.00E+08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
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