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You searched for: EV200159 (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
EV200159 2/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 89%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (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
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
Expi293F
Sample origin
CD63-mCherry
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Alix/ Flotillin1/ CD9/ mcherry
non-EV: Lamin B1
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
CD63-mCherry
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ mCherry/ Alix/ CD81
Not detected contaminants
Lamin B1
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
particles per milliliter of final volume of sample;Yes, other: 5,00E+13
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
56
EV200159 3/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 89%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (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
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
Expi293F
Sample origin
CD63-FLuc
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: CD63/ CD81/ Alix/ Flotillin1/ CD9
non-EV: Lamin B1
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
CD63-FLuc
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Western Blot
Detected EV-associated proteins
Flotillin1/ Alix/ Firely luciferase/ CD9/ CD63/ CD81
Not detected contaminants
Lamin B1
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
particles per milliliter of final volume of sample;Yes, other: 1,50E+13
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
80
EV200159 1/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 67%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide)
EV-METRIC
67% (92nd 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
Expi293F
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.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: Alix/ CD63/ Flotillin1/ CD9/ CD81
non-EV: Lamin B1
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
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)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD9/ CD63/ Alix/ CD81
Not detected contaminants
Lamin B1
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
No NA
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
56
EV200159 4/4 Homo sapiens Cell culture supernatant DG
(d)(U)C
Lázaro-Ibáñez, Elisa 2021 57%

Study summary

Full title
All authors
Elisa Lázaro-Ibáñez, Farid N Faruqu, Amer F Saleh, Andreia M Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T Al-Jamal, Niek Dekker
Journal
ACS Nano
Abstract
The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution (show more...)The ability to track extracellular vesicles (EVs) in vivo without influencing their biodistribution is a key requirement for their successful development as drug delivery vehicles and therapeutic agents. Here, we evaluated the effect of five different optical and nuclear tracers on the in vivo biodistribution of EVs. Expi293F EVs were labeled using either a noncovalent fluorescent dye DiR, or covalent modification with 111indium-DTPA, or bioengineered with fluorescent (mCherry) or bioluminescent (Firefly and NanoLuc luciferase) proteins fused to the EV marker, CD63. To focus specifically on the effect of the tracer, we compared EVs derived from the same cell source and administered systemically by the same route and at equal dose into tumor-bearing BALB/c mice. 111Indium and DiR were the most sensitive tracers for in vivo imaging of EVs, providing the most accurate quantification of vesicle biodistribution by ex vivo imaging of organs and analysis of tissue lysates. Specifically, NanoLuc fused to CD63 altered EV distribution, resulting in high accumulation in the lungs, demonstrating that genetic modification of EVs for tracking purposes may compromise their physiological biodistribution. Blood kinetic analysis revealed that EVs are rapidly cleared from the circulation with a half-life below 10 min. Our study demonstrates that radioactivity is the most accurate EV tracking approach for a complete quantitative biodistribution study including pharmacokinetic profiling. In conclusion, we provide a comprehensive comparison of fluorescent, bioluminescent, and radioactivity approaches, including dual labeling of EVs, to enable accurate spatiotemporal resolution of EV trafficking in mice, an essential step in developing EV therapeutics. (hide)
EV-METRIC
57% (87th 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
Expi293F
Sample origin
CD63-NLuc
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
Density gradient
(Differential) (ultra)centrifugation
Protein markers
EV: None
non-EV: None
Proteomics
no
EV density (g/ml)
1.10 - 1.13
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods/Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
CD63-NLuc
EV-producing cells
Expi293F
EV-harvesting Medium
Serum free medium
Cell viability
Yes
Cell viability (%)
Yes
Separation Method
Differential ultracentrifugation
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
Obtain an EV pellet :
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
9
Lowest density fraction
10%
Highest density fraction
50%
Total gradient volume, incl. sample (mL)
17
Sample volume (mL)
1
Orientation
Bottom-up
Rotor type
SW 32.1 Ti
Speed (g)
120000
Duration (min)
960
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
94
Pelleting: duration (min)
180
Pelleting: rotor type
Type 45 Ti
Pelleting: speed (g)
120000
Protein Concentration Method
Fluorometric assay (e.g. Qubit, NanoOrange,...)
Flow cytometry
Hardware adjustments
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
126-154
EV concentration
Yes
Particle yield
particles per milliliter of final volume of sample;Yes, other: 2,00E+13
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
Report size (nm)
75
1 - 4 of 4
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV200159
species
Homo sapiens
sample type
Cell culture
cell type
Expi293F
condition
CD63-mCherry
CD63-FLuc
Control condition
CD63-NLuc
separation protocol
Density gradient
dUC
Density gradient
dUC
Density gradient
dUC
Density gradient
dUC
Exp. nr.
2
3
1
4
EV-METRIC %
89
89
67
57