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You searched for: EV180051 (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.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV180051 2/2 Rattus norvegicus Blood plasma dUC Takov K 2018 66%

Study summary

Full title
All authors
Takov K, Yellon DM, Davidson SM
Journal
J Extracell Vesicles
Abstract
Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids (show more...)Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB+ lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium. (hide)
EV-METRIC
66% (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
Blood plasma
Sample origin
Control condition
Focus vesicles
small extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
dUC
Adj. k-factor
115.3 (pelleting) / 115.3 (washing)
Protein markers
EV: CD81/ HSP70/ Endothelin-1/ CD9
non-EV: ApoB
Proteomics
no
Show all info
Study aim
Function, Technical analysis comparing/optimizing EV-related methods, Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Pelleting: rotor type
MLA-55
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
115.3
Wash: time (min)
70
Wash: Rotor Type
MLA-55
Wash: speed (g)
100000
Wash: adjusted k-factor
115.3
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Concentration
13.6-14.6
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70
ELISA
Antibody details provided?
Yes
Other 1
Protein array (ARY007, R&D Systems)
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
96.6
EV concentration
Yes
Particle yield
20000000000
EM
EM-type
Transmission-EM
Image type
Wide-field
EV180051 1/2 Rattus norvegicus Blood plasma qEV
dUC
SEC
UF
UF
Takov K 2018 62%

Study summary

Full title
All authors
Takov K, Yellon DM, Davidson SM
Journal
J Extracell Vesicles
Abstract
Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids (show more...)Interest in small extracellular vesicles (sEVs) as functional carriers of proteins and nucleic acids is growing continuously. There are large numbers of sEVs in the blood, but lack of standardised methods for sEV isolation greatly limits our ability to study them. In this report, we use rat plasma to systematically compare two commonly used techniques for isolation of sEVs: ultracentrifugation (UC-sEVs) and size-exclusion chromatography (SEC-sEVs). SEC-sEVs had higher particle number, protein content, particle/protein ratios and sEV marker signal than UC-sEVs. However, SEC-sEVs also contained greater amounts of APOB+ lipoproteins and large quantities of non-sEV protein. sEV marker signal correlated very well with both particle number and protein content in UC-sEVs but not in all of the SEC-sEV fractions. Functionally, both UC-sEVs and SEC-sEVs isolates contained a variety of proangiogenic factors (with endothelin-1 being the most abundant) and stimulated migration of endothelial cells. However, there was no evident correlation between the promigratory potential and the quantity of sEVs added, indicating that non-vesicular co-isolates may contribute to the promigratory effects. Overall, our findings suggest that UC provides plasma sEVs of lower yields, but markedly higher purity compared to SEC. Furthermore, we show that the functional activity of sEVs can depend on the isolation method used and does not solely reflect the sEV quantity. These findings are of importance when working with sEVs isolated from plasma- or serum-containing conditioned medium. (hide)
EV-METRIC
62% (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
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
small extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
qEV + dUC + SEC + UF + UF
Protein markers
EV: CD81/ HSP70/ Endothelin-1/ CD9
non-EV: ApoB
Proteomics
no
Show all info
Study aim
Function, Technical analysis comparing/optimizing EV-related methods, Identification of content (omics approaches)
Sample
Species
Rattus norvegicus
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 10,000 g and 50,000 g
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Commercial kit
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Concentration
39-308
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
HSP70
ELISA
Antibody details provided?
Yes
Other 1
Protein array (ARY007, R&D Systems)
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
81.5
EV concentration
Yes
Particle yield
2000000000000
EM
EM-type
Transmission-EM
Image type
Wide-field
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV180051
species
Rattus norvegicus
sample type
Blood plasma
condition
Control condition
separation protocol
dUC
qEV
dUC
SEC
UF
UF
Exp. nr.
2
1
EV-METRIC %
66
62