Search > Results

You searched for: EV190046 (EV-TRACK ID)

Showing 1 - 2 of 2

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
EV190046 1/2 Homo sapiens Cell culture supernatant (d)(U)C Deville, Sarah 2021 56%

Study summary

Full title
All authors
Sarah Deville, Pascale Berckmans, Rebekka Van Hoof, Ivo Lambrichts, Anna Salvati, Inge Nelissen
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major (show more...)Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day. (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
THP-1
Sample origin
LPS-stimulated
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/ Flotillin1/ CD9
non-EV: Cytochrome C
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
LPS-stimulated
EV-producing cells
THP-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 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)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101
Not detected contaminants
Cytochrome C
Flow cytometry
Type of Flow cytometry
Hardware adjustments
Calibration bead size
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
182
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
A BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection was used for analysis. The device utilizes a highly sensitive fluorescence trigger to measure the EVs.
Calibration bead size
0.1
EV concentration
Yes
EV190046 2/2 Homo sapiens Cell culture supernatant exoEasy (Qiagen) Deville, Sarah 2021 50%

Study summary

Full title
All authors
Sarah Deville, Pascale Berckmans, Rebekka Van Hoof, Ivo Lambrichts, Anna Salvati, Inge Nelissen
Journal
PLoS One
Abstract
Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major (show more...)Extracellular vesicles (EVs) are of interest for a wide variety of biomedical applications. A major limitation for the clinical use of EVs is the lack of standardized methods for the fast and reproducible separation and subsequent detection of EV subpopulations from biofluids, as well as their storage. To advance this application area, fluorescence-based characterization technologies with single-EV resolution, such as high-sensitivity flow cytometry (HS-FCM), are powerful to allow assessment of EV fractionation methods and storage conditions. Furthermore, the use of HS-FCM and fluorescent labeling of EV subsets is expanding due to the potential of high-throughput, multiplex analysis, but requires further method development to enhance the reproducibility of measurements. In this study, we have applied HS-FCM measurements next to standard EV characterization techniques, including nanoparticle tracking analysis, to compare the yield and purity of EV fractions obtained from lipopolysaccharide-stimulated monocytic THP-1 cells by two EV isolation methods, differential centrifugation followed by ultracentrifugation and the exoEasy membrane affinity spin column purification. We observed differences in EV yield and purity. In addition, we have investigated the influence of EV storage at 4°C or -80°C for up to one month on the EV concentration and the stability of EV-associated fluorescent labels. The concentration of the in vitro cell derived EV fractions was shown to remain stable under the tested storage conditions, however, the fluorescence intensity of labeled EV stored at 4°C started to decline within one day. (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
THP-1
Sample origin
LPS-stimulated
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
exoEasy (Qiagen)
Protein markers
EV: / TSG101/ Flotillin1/ CD9
non-EV: Cytochrome C/ Cytochrome c
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
LPS-stimulated
EV-producing cells
THP-1
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Separation Method
Commercial kit
Other;exoEasy (Qiagen)
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ TSG101
Not detected contaminants
Cytochrome C
Flow cytometry
Type of Flow cytometry
Hardware adjustments
Calibration bead size
Detected EV-associated proteins
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
210
EV concentration
Yes
Particle analysis: flow cytometry
Flow cytometer type
BD Influx
Hardware adjustment
A BD Influx flow cytometer equipped with a high power 488-nm laser (200 mW) and a small-particle detector for high sensitivity forward scatter detection was used for analysis. The device utilizes a highly sensitive fluorescence trigger to measure the EVs.
Calibration bead size
0.1
Report type
Not Reported
EV concentration
Yes
1 - 2 of 2