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You searched for: EV140068 (EV-TRACK ID)

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Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, separation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Separation protocol
  • Gives a short, non-chronological overview of the different steps of the separation protocol.
    • (d)(U)C = (differential) (ultra)centrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
    • IAF = immuno-affinity capture
Experiment number
  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
Experiment number
  • Experiments differ in Isolation method
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV140068 1/3 Homo sapiens NAY (d)(U)C
Filtration
Cvjetkovic A 2014 44%

Study summary

Full title
All authors
Cvjetkovic A, Lötvall J, Lässer C
Journal
J Extracell Vesicles
Abstract
BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consi (show more...)BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consist of vesicle species ranging in size from 30 nm to 5 µm in diameter. These vesicles are most commonly isolated by differential centrifugations, which pellets particles based on their differential movement through the liquid medium in which they are immersed. Multiple parameters, including the utilization of different rotor types, can influence the yield and purity of isolated vesicles; however, the understanding of how these factors affect is limited. MATERIALS AND METHODS: Here, we compare the influence of multiple centrifugation parameters, including the use of swinging bucket and fixed angle rotors, as well as different centrifugation times, for the isolation of the smallest EVs, exosomes. In particular, we determine the yields of exosomal RNA and protein, as well as the nature of the isolated vesicles and possible protein contamination with methods such as electron microscopy, western blot and flow cytometry. RESULTS: Our results show that application of a specific g-force or rotation speed by itself does not predict the ability of pelleting exosomes, and that prolonged centrifugation times can achieve greater yields of exosomal RNA and protein, whereas very long centrifugation times result in excessive protein concentrations in the exosome pellet. CONCLUSION: In conclusion, rotor type, g-force and centrifugation times significantly influence exosome yield during centrifugation-based isolation procedures, and current commonly recommended isolation protocols may not be fully optimized for yield and purity of exosomes. (hide)
EV-METRIC
44% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
extracellular vesicles
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
(d)(U)C
Filtration
Adj. k-factor
132.9 (pelleting)
Protein markers
EV: Calnexin/ CD81/ TSG101/ CD63/ CD9
non-EV: Cell organelle protein
Proteomics
no
TEM measurements
53(median)
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
70Ti
Pelleting: adjusted k-factor
132.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
CD63/ CD81/ CD9/ TSG101/ Calnexin
Detected contaminants
Cell organelle protein
ELISA
Antibody details provided?
No
Detected EV-associated proteins
Calnexin
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
No
Selected surface protein(s)
Yes
EV140068 3/3 Homo sapiens NAY (d)(U)C
Filtration
Cvjetkovic A 2014 44%

Study summary

Full title
All authors
Cvjetkovic A, Lötvall J, Lässer C
Journal
J Extracell Vesicles
Abstract
BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consi (show more...)BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consist of vesicle species ranging in size from 30 nm to 5 µm in diameter. These vesicles are most commonly isolated by differential centrifugations, which pellets particles based on their differential movement through the liquid medium in which they are immersed. Multiple parameters, including the utilization of different rotor types, can influence the yield and purity of isolated vesicles; however, the understanding of how these factors affect is limited. MATERIALS AND METHODS: Here, we compare the influence of multiple centrifugation parameters, including the use of swinging bucket and fixed angle rotors, as well as different centrifugation times, for the isolation of the smallest EVs, exosomes. In particular, we determine the yields of exosomal RNA and protein, as well as the nature of the isolated vesicles and possible protein contamination with methods such as electron microscopy, western blot and flow cytometry. RESULTS: Our results show that application of a specific g-force or rotation speed by itself does not predict the ability of pelleting exosomes, and that prolonged centrifugation times can achieve greater yields of exosomal RNA and protein, whereas very long centrifugation times result in excessive protein concentrations in the exosome pellet. CONCLUSION: In conclusion, rotor type, g-force and centrifugation times significantly influence exosome yield during centrifugation-based isolation procedures, and current commonly recommended isolation protocols may not be fully optimized for yield and purity of exosomes. (hide)
EV-METRIC
44% (85th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods. For the quantitative method, the reporting of measured EV concentration is expected.
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
NAY
Focus vesicles
extracellular vesicles
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
(d)(U)C
Filtration
Adj. k-factor
216.9 (pelleting)
Protein markers
EV: Calnexin/ CD81/ TSG101/ CD63/ CD9
non-EV: Cell organelle protein
Proteomics
no
TEM measurements
53(median)
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
114
Pelleting: rotor type
SW32
Pelleting: adjusted k-factor
216.9
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Detected EV-associated proteins
CD63/ CD81/ CD9/ TSG101/ Calnexin
Detected contaminants
Cell organelle protein
ELISA
Antibody details provided?
No
Detected EV-associated proteins
Calnexin
Flow cytometry specific beads
Antibody details provided?
No
Antibody dilution provided?
No
Selected surface protein(s)
Yes
EV140068 2/3 Homo sapiens NAY (d)(U)C
Filtration
Cvjetkovic A 2014 14%

Study summary

Full title
All authors
Cvjetkovic A, Lötvall J, Lässer C
Journal
J Extracell Vesicles
Abstract
BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consi (show more...)BACKGROUND: Extracellular vesicles (EV), the collective term for vesicles released from cells, consist of vesicle species ranging in size from 30 nm to 5 µm in diameter. These vesicles are most commonly isolated by differential centrifugations, which pellets particles based on their differential movement through the liquid medium in which they are immersed. Multiple parameters, including the utilization of different rotor types, can influence the yield and purity of isolated vesicles; however, the understanding of how these factors affect is limited. MATERIALS AND METHODS: Here, we compare the influence of multiple centrifugation parameters, including the use of swinging bucket and fixed angle rotors, as well as different centrifugation times, for the isolation of the smallest EVs, exosomes. In particular, we determine the yields of exosomal RNA and protein, as well as the nature of the isolated vesicles and possible protein contamination with methods such as electron microscopy, western blot and flow cytometry. RESULTS: Our results show that application of a specific g-force or rotation speed by itself does not predict the ability of pelleting exosomes, and that prolonged centrifugation times can achieve greater yields of exosomal RNA and protein, whereas very long centrifugation times result in excessive protein concentrations in the exosome pellet. CONCLUSION: In conclusion, rotor type, g-force and centrifugation times significantly influence exosome yield during centrifugation-based isolation procedures, and current commonly recommended isolation protocols may not be fully optimized for yield and purity of exosomes. (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
NAY
Focus vesicles
extracellular vesicles
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
(d)(U)C
Filtration
Adj. k-factor
216.9 (pelleting)
Protein markers
EV:
non-EV:
Proteomics
no
Show all info
Study aim
Technical
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
EV Depleted
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
70
Pelleting: rotor type
SW32
Pelleting: adjusted k-factor
216.9
Filtration steps
0.22µm or 0.2µm
1 - 3 of 3
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV140068
species
Homo sapiens
sample type
Cell culture
cell type
NAY
condition
NAY
separation protocol
(d)(U)C
Filtration
(d)(U)C
Filtration
(d)(U)C
Filtration
Exp. nr.
1
3
2
EV-METRIC %
44
44
14