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You searched for: EV210035 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV210035 2/2 Homo sapiens Cell culture supernatant (d)(U)C
DG
Filtration
van Dommelen, Susan M 2016 78%

Study summary

Full title
All authors
Susan M van Dommelen, Roy van der Meel, Wouter W van Solinge, Maria Coimbra, Pieter Vader, Raymond M Schiffelers
Journal
Nanomedicine
Abstract
Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their co (show more...)Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their content reflects the parental cell status. This study aimed to examine whether tumor cell derived EVs mirrored the cellular changes caused by treatment with cetuximab, a therapeutic antibody that blocks activation of EGF receptor (EGFR). Materials & methods: A-431 cells were treated with cetuximab for 48 h. EVs were isolated using differential centrifugation and protein content was analyzed using western blotting. Results: EV levels of EGFR and phospho-EGFR were reduced after cetuximab treatment, reflecting similar changes in the parental cells. In addition, cetuximab was found associated with EVs. Conclusion: EVs could serve as biomarkers to monitor cetuximab treatment. Association of cetuximab with EVs might influence its behavior. Keywords: EGFR; biomarker; cancer therapy; cetuximab; diagnostics; exosomes; extracellular vesicles. (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
A-431
Sample origin
Cetuximab
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/ Alix/ CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ Cetuximab
non-EV: Lamin-A/ Lamin-C/ ATP5-A/ Tom20
Proteomics
no
EV density (g/ml)
1.10-1.21
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Cetuximab
EV-producing cells
A-431
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
JA-30.50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
JA-30.50
Wash: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4
Highest density fraction
2.5
Total gradient volume, incl. sample (mL)
Not specified
Sample volume (mL)
Not spec
Orientation
Top-down
Rotor type
SW 40 Ti
Speed (g)
200000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
4
Pelleting: duration (min)
70
Pelleting: rotor type
Not specified
Pelleting: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ Cetuximab/ TSG101/ Alix
Not detected contaminants
Lamin-A/ Lamin-C/ ATP5-A/ Tom20
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
EV210035 1/2 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
van Dommelen, Susan M 2016 56%

Study summary

Full title
All authors
Susan M van Dommelen, Roy van der Meel, Wouter W van Solinge, Maria Coimbra, Pieter Vader, Raymond M Schiffelers
Journal
Nanomedicine
Abstract
Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their co (show more...)Aim: Extracellular vesicles (EVs) are attractive candidates for biomarker research, because their content reflects the parental cell status. This study aimed to examine whether tumor cell derived EVs mirrored the cellular changes caused by treatment with cetuximab, a therapeutic antibody that blocks activation of EGF receptor (EGFR). Materials & methods: A-431 cells were treated with cetuximab for 48 h. EVs were isolated using differential centrifugation and protein content was analyzed using western blotting. Results: EV levels of EGFR and phospho-EGFR were reduced after cetuximab treatment, reflecting similar changes in the parental cells. In addition, cetuximab was found associated with EVs. Conclusion: EVs could serve as biomarkers to monitor cetuximab treatment. Association of cetuximab with EVs might influence its behavior. Keywords: EGFR; biomarker; cancer therapy; cetuximab; diagnostics; exosomes; extracellular vesicles. (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
A-431
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
Filtration
Protein markers
EV: TSG101/ Alix/ Cetuximab/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ CD9
non-EV: Lamin-A/ Lamin-C/ ATP5-A/ Tom20
Proteomics
no
Show all info
Study aim
Function/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
A-431
EV-harvesting Medium
Serum free medium
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
70
Pelleting: rotor type
JA-30.50
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
Not specified
Wash: time (min)
70
Wash: Rotor Type
JA-30.50
Wash: speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
CD9/ EGFR/ pEGFR/ Akt/ pAkt/ -actin/ TSG101/ Alix
Not detected EV-associated proteins
Cetuximab
Not detected contaminants
Lamin-A/ Lamin-C/ ATP5-A/ Tom20
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Not Reported
EV concentration
Yes
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