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You searched for: EV200110  (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
EV200110  1/3 Homo sapiens NCI-H1975 (d)(U)C
DG
Van Hoof R 2022 89%

Study summary

Full title
All authors
Van Hoof R, Deville S, Hollanders K, Berckmans P, Wagner P, Hooyberghs J, Nelissen I
Journal
Int J Mol Sci
Abstract
Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RN (show more...)Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (</ 200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor () gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (</ 35-100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. (hide)
EV-METRIC
89% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Density gradient
Protein markers
EV: CD81/ HSP70/ CD63/ CD9
non-EV: Calnexin/ Ribosomal protein S6
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
NCI-H1975
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: rotor type
SW 32.1 Ti
Pelleting: speed (g)
100000
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.7
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
366613
Duration (min)
960
Fraction volume (mL)
0.48
Fraction processing
None
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70/ CD81
Detected contaminants
Ribosomal protein S6
Not detected contaminants
Calnexin
Flow cytometry
Type of Flow cytometry
BD Influx flow cytometer
Hardware adaptation to ~100nm EV's
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
0.1/ 0.2
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)/ Other
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
Other/ RNase A/T1 mix
RNAse concentration
RNase A: 0.02 mg/ml - RNase T1: 50 U/ml
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
202
EV concentration
Yes
Particle yield
as number of particles per mililiter in the final EV fraction (500 l)/ other:: 5.00E+10
Particle analysis: flow cytometry
Flow cytometer type
BD Influx flow cytometer
Hardware adjustment
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
1
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200110  2/3 Homo sapiens NCI-H1975 (d)(U)C
UF
qEV
DG
Van Hoof R 2022 88%

Study summary

Full title
All authors
Van Hoof R, Deville S, Hollanders K, Berckmans P, Wagner P, Hooyberghs J, Nelissen I
Journal
Int J Mol Sci
Abstract
Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RN (show more...)Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (</ 200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor () gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (</ 35-100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Ultrafiltration
Commercial method
Density gradient
Protein markers
EV: CD81/ HSP70/ CD63/ CD9
non-EV: Calnexin/ Ribosomal protein S6
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
NCI-H1975
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.7
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
366613
Duration (min)
960
Fraction volume (mL)
0.48
Fraction processing
None
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Regenerated cellulose
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70/ CD81
Not detected contaminants
Calnexin/ Ribosomal protein S6
Flow cytometry
Type of Flow cytometry
BD Influx flow cytometer
Hardware adaptation to ~100nm EV's
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
0.1/ 0.2
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)/ Other
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
Other/ RNase A/T1 mix
RNAse concentration
RNase A: 0.02 mg/ml - RNase T1: 50 U/ml
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
186
EV concentration
Yes
Particle yield
as number of particles per mililiter in the final EV fraction (500 l)/ other:: 1.70E+10
Particle analysis: flow cytometry
Flow cytometer type
BD Influx flow cytometer
Hardware adjustment
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
1
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV200110  3/3 Homo sapiens NCI-H1975 (d)(U)C
Other/ exoEasy
Filtration
DG
Van Hoof R 2022 88%

Study summary

Full title
All authors
Van Hoof R, Deville S, Hollanders K, Berckmans P, Wagner P, Hooyberghs J, Nelissen I
Journal
Int J Mol Sci
Abstract
Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RN (show more...)Extracellular vesicles (EVs) are cell-derived structures surrounded by a lipid bilayer that carry RNA and DNA as potential templates for molecular diagnostics, e.g., in cancer genotyping. While it has been established that DNA templates appear on the outside of EVs, no consensus exists on which nucleic acid species inside small EVs (</ 200 nm, sEVs) are sufficiently abundant and accessible for developing genotyping protocols. We investigated this by extracting total intravesicular nucleic acid content from sEVs isolated from the conditioned cell medium of the human NCI-H1975 cell line containing the epidermal growth factor () gene mutation T790M as a model system for non-small cell lung cancer. We observed that mainly short genomic DNA (</ 35-100 bp) present in the sEVs served as a template. Using qEV size exclusion chromatography (SEC), significantly lower yield and higher purity of isolated sEV fractions were obtained as compared to exoEasy membrane affinity purification and ultracentrifugation. Nevertheless, we detected the T790M mutation in the sEVs' lumen with similar sensitivity using digital PCR. When applying SEC-based sEV separation prior to cell-free DNA extraction on spiked human plasma samples, we found significantly higher mutant allele frequencies as compared to standard cell-free DNA extraction, which in part was due to co-purification of circulating tumor DNA. We conclude that intravesicular genomic DNA can be exploited next to ctDNA to enhance T790M mutation detection sensitivity by adding a fast and easy-to-use sEV separation method, such as SEC, upstream of standard clinical cell-free DNA workflows. (hide)
EV-METRIC
88% (98th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Commercial method
Filtration
Density gradient
Protein markers
EV: CD81/ HSP70/ CD63/ CD9
non-EV: Calnexin/ Ribosomal protein S6
Proteomics
no
Show all info
Study aim
Biomarker/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
NCI-H1975
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Pelleting performed
No
Density gradient
Only used for validation of main results
Yes
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
10%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
4.7
Sample volume (mL)
0.3
Orientation
Bottom-up
Rotor type
SW 55 Ti
Speed (g)
366613
Duration (min)
960
Fraction volume (mL)
0.48
Fraction processing
None
Filtration steps
> 0.45 µm, 0.22µm or 0.2µm
Commercial kit
Other/ exoEasy
Characterization: Protein analysis
Protein Concentration Method
Lowry-based assay
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70/ CD81
Not detected contaminants
Calnexin/ Ribosomal protein S6
Flow cytometry
Type of Flow cytometry
BD Influx flow cytometer
Hardware adaptation to ~100nm EV's
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
0.1/ 0.2
Antibody details provided?
No
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: RNA analysis
RNA analysis
Type
(RT)(q)PCR/ Capillary electrophoresis (e.g. Bioanalyzer)/ Other
Database
Yes
Proteinase treatment
No
RNAse treatment
Yes
Moment of RNAse treatment
After
RNAse type
Other/ RNase A/T1 mix
RNAse concentration
RNase A: 0.02 mg/ml - RNase T1: 50 U/ml
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
250
EV concentration
Yes
Particle yield
as number of particles per mililiter in the final EV fraction (500 l)/ other:: 2.60E+11
Particle analysis: flow cytometry
Flow cytometer type
BD Influx flow cytometer
Hardware adjustment
EV samples were analyzed using a BD Influx flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) equipped with a 488 nm high power laser (200 mW) and a small-particle detector as previously described by van der Vlist et al. (2012).
Calibration bead size
1
Report type
Not Reported
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
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