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You searched for: EV210237 (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
EV210237 1/2 Homo sapiens Blood plasma qEV Gelibter S 2022 75%

Study summary

Full title
All authors
Gelibter S, Marostica G, Mandelli A, Siciliani S, Podini P, Finardi A, Furlan R
Journal
J Extracell Vesicles
Abstract
Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. Wh (show more...)Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While -80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of -80°C storage and freeze-thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment/ flow cytometry for the analysis of two single fluorescent-tagged EVs populations (GFP and mCherry), mixed before preservation. We found that -80°C storage reduces EVs concentration and sample purity in a time-dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size-charge plots. None of the tested conditions prevented the observed effects. Freeze-thaw cycles lead to an EVs reduction after the first cycle and to a cycle-dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double-positive EVs (GFP -mCherry ). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non-archival, samples in majority of cases. (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
qEV
Protein markers
EV: Alix/ Flotillin-1/ Lamp1/ ANXA1
non-EV: GM130/ ApoE/ H3
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
particles per milliliter of starting sample
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ Flotillin-1/ Lamp1/ ANXA1
Not detected contaminants
GM130/ ApoE/ H3
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
50-350
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.00e+11
EM
EM-type
Immuno-EM
EM protein
CD63
Image type
Wide-field
EV210237 2/2 Mus musculus Cell culture supernatant (d)(U)C Gelibter S 2022 56%

Study summary

Full title
All authors
Gelibter S, Marostica G, Mandelli A, Siciliani S, Podini P, Finardi A, Furlan R
Journal
J Extracell Vesicles
Abstract
Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. Wh (show more...)Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While -80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of -80°C storage and freeze-thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment/ flow cytometry for the analysis of two single fluorescent-tagged EVs populations (GFP and mCherry), mixed before preservation. We found that -80°C storage reduces EVs concentration and sample purity in a time-dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size-charge plots. None of the tested conditions prevented the observed effects. Freeze-thaw cycles lead to an EVs reduction after the first cycle and to a cycle-dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double-positive EVs (GFP -mCherry ). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non-archival, samples in majority of cases. (hide)
EV-METRIC
56% (85th 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
NA
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
Protein markers
EV: Alix/ Flotillin-1/ Lamp1/ ANXA1/ IB4
non-EV: GM130/ H3
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
BV2
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell count
40000000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
4h
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Wash: volume per pellet (ml)
12
Wash: time (min)
960
Wash: Rotor Type
SW 41 Ti
Wash: speed (g)
100000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix/ Flotillin-1/ Lamp1/ ANXA1
Detected contaminants
H3
Not detected contaminants
GM130
Flow cytometry
Type of Flow cytometry
CytoflexS
Calibration bead size
0.1, 0.3, 0.5, 0.7, 0.9
Antibody details provided?
Yes
Detected EV-associated proteins
IB4
Characterization: Lipid analysis
No
Characterization: Particle analysis
TRPS
Report type
Mean
Reported size (nm)
50-350
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.00e+11
Particle analysis: flow cytometry
Flow cytometer type
CytoflexS
Hardware adjustment
Calibration bead size
0.1, 0.3, 0.5, 0.7, 0.9
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.00e+7
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV210237
species
Homo sapiens
Mus musculus
sample type
Blood plasma
Cell culture
cell type
NA
BV2
medium
NA
EV-depleted medium
condition
Control condition
NA
separation protocol
qEV
dUC
Exp. nr.
1
2
EV-METRIC %
75
56