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You searched for: EV200093 (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
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV200093 10/10 Homo sapiens Blood plasma (d)(U)C
Filtration
DG
Dong, Liang 2021 63%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
63% (93rd 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
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
(d)(U)C
Filtration
DG
Protein markers
EV: CD81/ Flotillin1
non-EV: ApoA1
Proteomics
no
EV density (g/ml)
1.10-1.15
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
38
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
230
Fraction volume (mL)
4.75
Fraction processing
Centrifugation
Pelleting: volume per fraction
28
Pelleting: duration (min)
60
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD81
Not detected contaminants
ApoA1
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Not Reported
EV200093 1/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Dong, Liang 2021 56%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (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
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
PC3
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
(d)(U)C
Filtration
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
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 70 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
28
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
120000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
113.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 9.05E+07
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
EV concentration
Yes
EV200093 6/10 Homo sapiens Blood plasma (d)(U)C
Filtration
Dong, Liang 2021 56%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
56% (90th 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
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
(d)(U)C
Filtration
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: ApoA1/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
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 70 Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
28
Wash: time (min)
120
Wash: Rotor Type
Type 70 Ti
Wash: speed (g)
120000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD81
Detected contaminants
ApoA1
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
121.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.42E+09
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
58.25
EV concentration
Yes
EV200093 2/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Total Exosome Isolation
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (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
Cell Name
PC3
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
(d)(U)C
Filtration
Total Exosome Isolation
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
110.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 4.42E+08
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
59.75
EV concentration
Yes
EV200093 3/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
Exodisc
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (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
Cell Name
PC3
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
(d)(U)C
Filtration
Exodisc
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
115.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.19E+08
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
57.25
EV concentration
Yes
EV200093 4/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
SEC and UF
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (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
Cell Name
PC3
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
(d)(U)C
Filtration
SEC and UF
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
microBCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD63/ CD81
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
114.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 6.73E+07
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
59.25
EV concentration
Yes
EV200093 5/10 Homo sapiens Cell culture supernatant (d)(U)C
Filtration
DG
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (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
Cell Name
PC3
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
(d)(U)C
Filtration
DG
Protein markers
EV: CD81/ Flotillin1
non-EV: None
Proteomics
no
EV density (g/ml)
1.10-1.15
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
PC3
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability
Yes
Cell viability (%)
Yes
Cell number specification
Yes
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Continuous
Lowest density fraction
5%
Highest density fraction
60%
Total gradient volume, incl. sample (mL)
38
Sample volume (mL)
2
Orientation
Bottom-up
Rotor type
SW 32 Ti
Speed (g)
100000
Duration (min)
230
Fraction volume (mL)
4.75
Fraction processing
Centrifugation
Pelleting: volume per fraction
28
Pelleting: duration (min)
60
Pelleting: rotor type
Type 70 Ti
Pelleting: speed (g)
120000
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Flotillin1/ CD81
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Characterization: Particle analysis
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
EV200093 7/10 Homo sapiens Blood plasma (d)(U)C
Filtration
Total Exosome Isolation
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (84th 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
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
(d)(U)C
Filtration
Total Exosome Isolation
Protein markers
EV: CD81/ Flotillin1/ None/ CD63/ CD9
non-EV: ApoA1/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Commercial kit
Total Exosome Isolation
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
None
Not detected EV-associated proteins
CD81/ Flotillin1
Detected contaminants
ApoA1
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
None
Not detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
127.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.70E+11
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
61.75
EV concentration
Yes
EV200093 8/10 Homo sapiens Blood plasma (d)(U)C
Filtration
Exodisc
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (84th 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
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
(d)(U)C
Filtration
Exodisc
Protein markers
EV: CD81/ Flotillin1/ None/ CD63/ CD9
non-EV: ApoA1/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
None
Not detected EV-associated proteins
CD81/ Flotillin1
Detected contaminants
ApoA1
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
None
Not detected EV-associated proteins
CD9/ CD63/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
129.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 2.29E+11
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
62.25
EV concentration
Yes
EV200093 9/10 Homo sapiens Blood plasma (d)(U)C
Filtration
SEC and UF
Dong, Liang 2021 50%

Study summary

Full title
All authors
Liang Dong, Richard C. Zieren, Kengo Horie, Chi‐Ju Kim, Emily Mallick, Yuezhou Jing, Mingxiao Feng, Morgan D. Kuczler, Jordan Green, Sarah R. Amend, Kenneth W. Witwer, Theo M. de Reijke, Yoon‐Kyoung Cho, Kenneth J. Pienta, Wei Xue
Journal
J Extracell Vesicles
Abstract
One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the l (show more...)One of the challenges that restricts the evolving extracellular vesicle (EV) research field is the lack of a consensus method for EV separation. This may also explain the diversity of the experimental results, as co‐separated soluble proteins and lipoproteins may impede the interpretation of experimental findings. In this study, we comprehensively evaluated the EV yields and sample purities of three most popular EV separation methods, ultracentrifugation, precipitation and size exclusion chromatography combined with ultrafiltration, along with a microfluidic tangential flow filtration device, Exodisc, in three commonly used biological samples, cell culture medium, human urine and plasma. Single EV phenotyping and density‐gradient ultracentrifugation were used to understand the proportion of true EVs in particle separations. Our findings suggest Exodisc has the best EV yield though it may co‐separate contaminants when the non‐EV particle levels are high in input materials. We found no 100% pure EV preparations due to the overlap of their size and density with many non‐EV particles in biofluids. Precipitation has the lowest sample purity, regardless of sample type. The purities of the other techniques may vary in different sample types and are largely dependent on their working principles and the intrinsic composition of the input sample. Researchers should choose the proper separation method according to the sample type, downstream analysis and their working scenarios. (hide)
EV-METRIC
50% (84th 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
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
(d)(U)C
Filtration
SEC and UF
Protein markers
EV: CD81/ Flotillin1/ CD63/ CD9
non-EV: ApoA1/ Calreticulin
Proteomics
no
Show all info
Study aim
Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Blood plasma
Sample Condition
Control condition
Separation Method
Differential ultracentrifugation
centrifugation steps
Between 800 g and 10,000 g
Between 10,000 g and 50,000 g
Filtration steps
0.45µm > x > 0.22µm,
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Detected EV-associated proteins
Flotillin1/ CD81
Detected contaminants
ApoA1
Not detected contaminants
Calreticulin
Flow cytometry
Type of Flow cytometry
NanoFCM
Hardware adjustments
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanopar
Calibration bead size
0.2
Detected EV-associated proteins
CD63/ CD9/ CD81
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
128.5
EV concentration
Yes
Particle yield
Yes, as number of particles per milliliter of starting sample 1.05E+11
Particle analysis: flow cytometry
Flow cytometer type
NanoFCM
Hardware adjustment
Please refer to the publication below: Zhu S, Ma L, Wang S, et al. Light-scattering detection below the level of single fluorescent molecules for high-resolution characterization of functional nanoparticles. ACS nano. 2014 Oct 28;8(10):10998-1006.
Calibration bead size
0.2
Report type
Modus
Reported size (nm)
58.25
EV concentration
Yes
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