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You searched for: EV220003 (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 code Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV220003 1/2 Homo sapiens Blood plasma DG
SEC (non-commercial)
Karimi, Nasibeh 2022 75%

Study summary

Full title
All authors
Nasibeh Karimi, Razieh Dalirfardouei, Tomás Dias, Jan Lötvall, Cecilia Lässer
Journal
J Extracell Vesicles
Abstract
Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the developme (show more...)Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail. (hide)
EV-METRIC
75% (97th 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
Blood plasma
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
Density gradient
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin
non-EV: Calnexin
Proteomics
no
EV density (g/ml)
1.06-1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
12 ml
Sample volume (mL)
6 ml
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
178,000
Duration (min)
180
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
27.8
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin
Not detected contaminants
Calnexin
Detected EV-associated proteins
CD9/ CD63/ CD81/ CD41a
Detected EV-associated proteins
CD9/ CD63/ CD81/ CD41a
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
as total number of particles in pooled SEC fractions from 6 mL of sample: 2.90e+9
Report type
Size range/distribution
Report size
50-70
EV-concentration
No
EV220003 2/2 Homo sapiens Serum DG
SEC (non-commercial)
Karimi, Nasibeh 2022 63%

Study summary

Full title
All authors
Nasibeh Karimi, Razieh Dalirfardouei, Tomás Dias, Jan Lötvall, Cecilia Lässer
Journal
J Extracell Vesicles
Abstract
Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the developme (show more...)Background: The ability to isolate extracellular vesicles (EVs) from blood is vital in the development of EVs as disease biomarkers. Both serum and plasma can be used, but few studies have compared these sources in terms of the type of EVs that are obtained. The aim of this study was to determine the presence of different subpopulations of EVs in plasma and serum. Method: Blood was collected from healthy subjects, and plasma and serum were isolated in parallel. ACD or EDTA tubes were used for the collection of plasma, while serum was obtained in clot activator tubes. EVs were isolated utilising a combination of density cushion and SEC, a combination of density cushion and gradient or by a bead antibody capturing system (anti-CD63, anti-CD9 and anti-CD81 beads). The subpopulations of EVs were analysed by NTA, Western blot, SP-IRIS, conventional and nano flow cytometry, magnetic bead ELISA and mass spectrometry. Additionally, different isolation protocols for plasma were compared to determine the contribution of residual platelets in the analysis. Results: This study shows that a higher number of CD9+ EVs were present in EDTA-plasma compared to ACD-plasma and to serum, and the presence of CD41a on these EVs suggests that they were released from platelets. Furthermore, only a very small number of EVs in blood were double-positive for CD63 and CD81. The CD63+ EVs were enriched in serum, while CD81+ vesicles were the rarest subpopulation in both plasma and serum. Additionally, EDTA-plasma contained more residual platelets than ACD-plasma and serum, and two centrifugation steps were crucial to reduce the number of platelets in plasma prior to EV isolation. Conclusion: These results show that human blood contains multiple subpopulations of EVs that carry different tetraspanins. Blood sampling methods, including the use of anti-coagulants and choice of centrifugation protocols, can affect EV analyses and should always be reported in detail. (hide)
EV-METRIC
63% (95th 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
Serum
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
Density gradient
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ CD63/ CD81/ Flotillin-1/ CD41a/ P-selectin
non-EV: None
Proteomics
no
EV density (g/ml)
1.06-1.16
Show all info
Study aim
Biomarker/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Serum
Separation Method
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
3
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
12 ml
Sample volume (mL)
6 ml
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
178,000
Duration (min)
180
Fraction volume (mL)
1
Fraction processing
Size-exclusion chromatography
Size-exclusion chromatography
Total column volume (mL)
10
Sample volume/column (mL)
1
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay
Protein Yield (µg)
26.7
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD9/ CD63/ CD81
Not detected EV-associated proteins
Flotillin-1/ CD41a/ P-selectin
Detected EV-associated proteins
CD9/ CD63/ CD81/ CD41a
Detected EV-associated proteins
CD9/ CD63/ CD81/ CD41a
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
EV concentration
Yes
Particle yield
as total number of particles in pooled SEC fractions from 6 mL of sample: 2.56e+9
Report type
Size range/distribution
Report size
50-70
EV-concentration
No
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV220003
species
Homo sapiens
sample type
Blood plasma
Serum
condition
Control condition
Control condition
separation protocol
Density gradient/
Size-exclusion chromatography
(non-commercial)
Density gradient/
Size-exclusion chromatography
(non-commercial)
Exp. nr.
1
2
EV-METRIC %
75
63