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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
Experiment number
  • Experiments differ in Sample type/Isolation method
Experiment number
  • Experiments differ in Sample type/Isolation method
Experiment number
  • Experiments differ in Sample type/Isolation method
Experiment number
  • Experiments differ in Sample type/Isolation method
Experiment number
  • Experiments differ in Sample type/Isolation method
Experiment number
  • Experiments differ in Sample type/Isolation method
Experiment number
  • Experiments differ in Sample type/Isolation method
Experiment number
  • Experiments differ in Sample type/Isolation method
Experiment number
  • Experiments differ in Sample type/Isolation method
Experiment number
  • Experiments differ in Sample type/Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV150007 1/10 Homo sapiens Cell culture supernatant (d)(U)C
qEV
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (81st 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
Sample origin
DNF
Focus vesicles
exosomes
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
(d)(U)C + qEV
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Commercial kit
qEV
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV150007 4/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (81st 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
Sample origin
DNF
Focus vesicles
exosomes
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
(d)(U)C + Filtration
Protein markers
EV: TSG101/ HSP70/ Flotilin1/ CD63
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV150007 5/10 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (81st 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
Sample origin
DNF
Focus vesicles
exosomes
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
DG + (d)(U)C + Filtration
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Density gradient
Density medium
Iodixanol
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Rotor type
50.2Ti
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EV150007 7/10 Homo sapiens Cell culture supernatant (d)(U)C
Exo-spin
Filtration
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (81st 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
Sample origin
DNF
Focus vesicles
exosomes
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
(d)(U)C + Exo-spin + Filtration
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
Exo-spin
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Close-up
EV150007 8/10 Homo sapiens Cell culture supernatant (d)(U)C
ExoQuick
Filtration
Lobb RJ 2015 50%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
50% (81st 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
Sample origin
DNF
Focus vesicles
exosomes
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
(d)(U)C + ExoQuick + Filtration
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Detected contaminants
Cell organelle protein
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Close-up
EV150007 3/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Lobb RJ 2015 44%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
44% (76th 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
Sample origin
DNF
Focus vesicles
exosomes
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
(d)(U)C + Filtration
Adj. k-factor
157.1 (pelleting) / 157.1 (washing)
Protein markers
EV: TSG101/ HSP70/ Flotilin1
non-EV:
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
50.2Ti
Pelleting: adjusted k-factor
157.1
Wash: volume per pellet (ml)
1
Wash: Rotor Type
50.2Ti
Wash: adjusted k-factor
157.1
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ HSP70/ TSG101
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV150007 2/10 Homo sapiens Blood plasma (d)(U)C
qEV
Lobb RJ 2015 38%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
38% (74th 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
DNF
Focus vesicles
exosomes
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
(d)(U)C + qEV
Protein markers
EV: Flotilin1
non-EV: Albumin/ Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Commercial kit
qEV
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1
Detected contaminants
Cell organelle protein/ Albumin
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV150007 9/10 Homo sapiens Blood plasma (d)(U)C
ExoQuick
Filtration
Lobb RJ 2015 38%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
38% (74th 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
DNF
Focus vesicles
exosomes
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
(d)(U)C + ExoQuick + Filtration
Protein markers
EV: Flotilin1
non-EV: Albumin/ Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
ExoQuick
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1
Detected contaminants
Cell organelle protein/ Albumin
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Close-up
EV150007 10/10 Homo sapiens Blood plasma (d)(U)C
Exo-spin
Filtration
Lobb RJ 2015 38%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
38% (74th 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
DNF
Focus vesicles
exosomes
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
(d)(U)C + Exo-spin + Filtration
Protein markers
EV: Flotilin1
non-EV: Albumin/ Cell organelle protein
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.22µm or 0.2µm
Commercial kit
Exo-spin
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1
Detected contaminants
Cell organelle protein/ Albumin
Characterization: Particle analysis
TRPS
EM
EM-type
transmission EM
Image type
Wide-field
EV150007 6/10 Homo sapiens Cell culture supernatant DG
(d)(U)C
Filtration
Lobb RJ 2015 33%

Study summary

Full title
All authors
Lobb RJ, Becker M, Wen SW, Wong CS, Wiegmans AP, Leimgruber A, Möller A
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of (show more...)Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research. (hide)
EV-METRIC
33% (62nd 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
Sample origin
DNF
Focus vesicles
exosomes
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
DG + (d)(U)C + Filtration
Adj. k-factor
157.1 (pelleting) / 157.1 (washing)
Protein markers
EV: TSG101/ Flotilin1
non-EV:
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Separation Method
Differential ultracentrifugation
centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Obtain an EV pellet :
Yes
Pelleting: time(min)
90
Pelleting: rotor type
50.2Ti
Pelleting: adjusted k-factor
157.1
Wash: volume per pellet (ml)
1
Wash: Rotor Type
50.2Ti
Wash: adjusted k-factor
157.1
Density gradient
Density medium
Iodixanol
Lowest density fraction
5
Highest density fraction
40
Orientation
Top-down
Rotor type
50.2Ti
Speed (g)
100000
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotilin1/ TSG101
Characterization: Particle analysis
TRPS
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