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You searched for: EV220091 (EV-TRACK ID)

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Results list Study Tree
Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV220091 1/8 Homo sapiens HeLa (d)(U)C
SEC (non-commercial) 		Visan, Kekoolani 2022 67%

Study summary

Full title
Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles
All authors
Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (hide)
EV-METRIC
67% (94th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ HSP70/ TSG101
non-EV: Calnexin/ Albumin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HeLa
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
1
Wash: time (min)
90
Wash: Rotor Type
S55-A2
Wash: speed (g)
100,000
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Detected EV-associated proteins
CD9/ HSP70/ TSG101
Detected contaminants
Albumin
Not detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
117.7
EV concentration
Yes
Particle yield
Total Particles: 1.63E+09
TRPS
Report type
Modus
Reported size (nm)
105.3333333
EV concentration
Yes
Particle yield
Total Particles: 8.91E+07
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220091 2/8 Homo sapiens HeLa UF
SEC (non-commercial) 		Visan, Kekoolani 2022 63%

Study summary

Full title
Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles
All authors
Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ Flotillin-1/ HSP70/ TSG101
non-EV: Calnexin/ Albumin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
HeLa
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Ultra filtration
Cut-off size (kDa)
300
Membrane type
Polyethersulfone (PES)
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Detected EV-associated proteins
CD9/ Flotillin-1/ HSP70/ TSG101
Detected contaminants
Albumin
Not detected contaminants
Calnexin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
133
EV concentration
Yes
Particle yield
Total Particles: 4.45E+10
TRPS
Report type
Modus
Reported size (nm)
98.33333333
EV concentration
Yes
Particle yield
Total Particles: 9.01E+08
EM
EM-type
Transmission-EM
Image type
Close-up, Wide-field
EV220091 6/8 Mus musculus EO771 UF
SEC (non-commercial) 		Visan, Kekoolani 2022 38%

Study summary

Full title
Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles
All authors
Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (hide)
EV-METRIC
38% (79th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ HSP70/ CD63
non-EV: Albumin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
EO771
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Ultra filtration
Cut-off size (kDa)
300
Membrane type
Polyethersulfone (PES)
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Detected EV-associated proteins
CD9/ HSP70
Detected contaminants
Albumin
Flow cytometry
Type of Flow cytometry
Amnis® ImageStream®X Mark II Imaging Flow Cytometer
Calibration bead size
0.8
Detected EV-associated proteins
CD9/ CD63
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
142.9
EV concentration
Yes
Particle yield
Total Particles: 1.40333E+11
EV220091 5/8 Mus musculus EO771 (d)(U)C
SEC (non-commercial) 		Visan, Kekoolani 2022 33%

Study summary

Full title
Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles
All authors
Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (hide)
EV-METRIC
33% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ HSP70
non-EV: Albumin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
EO771
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
100,000
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Detected EV-associated proteins
CD9/ HSP70
Detected contaminants
Albumin
Flow cytometry
Type of Flow cytometry
Amnis® ImageStream®X Mark II Imaging Flow Cytometer
Calibration bead size
0.8
Detected EV-associated proteins
CD9
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
129.2
EV concentration
Yes
Particle yield
Total Particles: 27900000000
EV220091 7/8 Mus musculus B16F10 (d)(U)C
SEC (non-commercial) 		Visan, Kekoolani 2022 33%

Study summary

Full title
Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles
All authors
Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (hide)
EV-METRIC
33% (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. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ HSP70
non-EV: Albumin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
B16F10
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
100,000
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Detected EV-associated proteins
CD9/ HSP70
Detected contaminants
Albumin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
137.5
EV concentration
Yes
Particle yield
Total Particles: 9205000000
EV220091 8/8 Mus musculus B16F10 UF
SEC (non-commercial) 		Visan, Kekoolani 2022 25%

Study summary

Full title
Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles
All authors
Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (hide)
EV-METRIC
25% (63rd percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: CD9/ HSP70
non-EV: Albumin
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
B16F10
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Ultra filtration
Cut-off size (kDa)
300
Membrane type
Polyethersulfone (PES)
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Characterization: Protein analysis
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Western Blot
Detected EV-associated proteins
CD9/ HSP70
Detected contaminants
Albumin
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
125.8
EV concentration
Yes
Particle yield
Total Particles: 2.09269E+11
EV220091 3/8 Homo sapiens MDAMB231 (d)(U)C
SEC (non-commercial) 		Visan, Kekoolani 2022 14%

Study summary

Full title
Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles
All authors
Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (hide)
EV-METRIC
14% (44th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
(Differential) (ultra)centrifugation
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDAMB231
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
120
Pelleting: rotor type
Type 50.2 Ti
Pelleting: speed (g)
100,000
Wash: volume per pellet (ml)
1
Wash: time (min)
120
Wash: Rotor Type
Type 50.2 Ti
Wash: speed (g)
100,000
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Characterization: Protein analysis
None
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
136.2555556
EV concentration
Yes
Particle yield
Total Particles: 1.88E+09
EV220091 4/8 Homo sapiens MDAMB231 UF
SEC (non-commercial) 		Visan, Kekoolani 2022 0%

Study summary

Full title
Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles
All authors
Kekoolani S. Visan, Richard J. Lobb, Sunyoung Ham, Luize G. Lima, Carlos Palma, Chai Pei Zhi Edna, Li-Ying Wu, Harsha Gowda, Keshava K. Datta, Gunter Hartel, Carlos Salomon, Andreas Möller
Journal
J Extracell Vesicles
Abstract
Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, progno (show more...)Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum-containing media. The majority of researchers use serum-containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non-sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA-MB-231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano-flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large-scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations. (hide)
EV-METRIC
0% (median: 14% of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Control condition
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = (Differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Ultrafiltration
Size-exclusion chromatography (non-commercial)
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
New methodological development/Technical analysis comparing/optimizing EV-related methods
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
MDAMB231
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
overnight (16h) at >=100,000g
Separation Method
Ultra filtration
Cut-off size (kDa)
300
Membrane type
Polyethersulfone (PES)
Size-exclusion chromatography
Used for validation?
Yes
Total column volume (mL)
10
Sample volume/column (mL)
0.5
Characterization: Protein analysis
None
Protein Concentration Method
Bradford
Protein Yield (µg)
per milliliter of starting sample
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
153.7
EV concentration
Yes
Particle yield
Total Particles: 36921301826
1 - 8 of 8
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV220091
species
Homo
sapiens
Homo
sapiens
Mus
musculus
Mus
musculus
Mus
musculus
Mus
musculus
Homo
sapiens
Homo
sapiens
sample type
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
cell type
HeLa
HeLa
EO771
EO771
B16F10
B16F10
MDAMB231
MDAMB231
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
Control
condition
separation protocol
dUC/
Size-exclusion
chromatography
(non-commercial)
Ultrafiltration/
Size-exclusion
chromatography
(non-commercial)
Ultrafiltration/
Size-exclusion
chromatography
(non-commercial)
dUC/
Size-exclusion
chromatography
(non-commercial)
dUC/
Size-exclusion
chromatography
(non-commercial)
Ultrafiltration/
Size-exclusion
chromatography
(non-commercial)
dUC/
Size-exclusion
chromatography
(non-commercial)
Ultrafiltration/
Size-exclusion
chromatography
(non-commercial)
Exp. nr.
1
2
6
5
7
8
3
4
EV-METRIC %
67
63
38
33
33
25
14
0