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You searched for: EV190019 (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.
    • dUC = (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
EV190019 1/4 Mus musculus Cell culture supernatant DG
dUC
Leermakers PA 2019 44%

Study summary

Full title
All authors
Leermakers PA, Remels AHV, Zonneveld MI, Rouschop KMA, Schols AMWJ, Gosker HR.
Journal
FASEB J
Abstract
Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated wi (show more...)Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes. (hide)
EV-METRIC
44% (78th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
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
DG + dUC
Protein markers
EV: HSP70/ Flotillin1
non-EV:
Proteomics
no
EV density (g/ml)
1.12-1.19 g/ml
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
C2C12
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 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12.4
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
188000
Duration (min)
16
Fraction volume (mL)
1 ml
Fraction processing
Centrifugation
Pelleting: volume per fraction
6 ml
Pelleting: duration (min)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
sedimentation speed;Other
Used subtypes
16,000g
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin1/ HSP70
NA
EM
EM-type
Cryo-EM
Image type
Wide-field
EV190019 2/4 Mus musculus Cell culture supernatant DG
dUC
Leermakers PA 2019 44%

Study summary

Full title
All authors
Leermakers PA, Remels AHV, Zonneveld MI, Rouschop KMA, Schols AMWJ, Gosker HR.
Journal
FASEB J
Abstract
Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated wi (show more...)Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes. (hide)
EV-METRIC
44% (78th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
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
DG + dUC
Protein markers
EV: HSP70/ Flotillin1
non-EV:
Proteomics
no
EV density (g/ml)
1.12-1.19 g/ml
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Control condition
EV-producing cells
C2C12
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 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12.4
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
188000
Duration (min)
16
Fraction volume (mL)
1 ml
Fraction processing
Centrifugation
Pelleting: volume per fraction
6 ml
Pelleting: duration (min)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
sedimentation speed;Other
Used subtypes
100,000g
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin1/ HSP70
NA
EM
EM-type
Cryo-EM
Image type
Wide-field
EV190019 3/4 Mus musculus Cell culture supernatant DG
dUC
Leermakers PA 2019 44%

Study summary

Full title
All authors
Leermakers PA, Remels AHV, Zonneveld MI, Rouschop KMA, Schols AMWJ, Gosker HR.
Journal
FASEB J
Abstract
Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated wi (show more...)Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes. (hide)
EV-METRIC
44% (78th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Deferiprone/iron chelation
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
DG + dUC
Protein markers
EV: HSP70/ Flotillin1
non-EV:
Proteomics
no
EV density (g/ml)
1.12-1.19 g/ml
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Deferiprone/iron chelation
EV-producing cells
C2C12
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 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12.4
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
188000
Duration (min)
16
Fraction volume (mL)
1 ml
Fraction processing
Centrifugation
Pelleting: volume per fraction
6 ml
Pelleting: duration (min)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
sedimentation speed;Other
Used subtypes
16,000g
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin1/ HSP70
NA
EM
EM-type
Cryo-EM
Image type
Wide-field
EV190019 4/4 Mus musculus Cell culture supernatant DG
dUC
Leermakers PA 2019 44%

Study summary

Full title
All authors
Leermakers PA, Remels AHV, Zonneveld MI, Rouschop KMA, Schols AMWJ, Gosker HR.
Journal
FASEB J
Abstract
Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated wi (show more...)Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes. (hide)
EV-METRIC
44% (78th percentile of all experiments on the same sample type)
 Reported
 Not reported
 Not applicable
EV-enriched proteins
Protein analysis: analysis of three or more EV-enriched proteins
non EV-enriched protein
Protein analysis: assessment of a non-EV-enriched protein
qualitative and quantitative analysis
Particle analysis: implementation of both qualitative and quantitative methods
electron microscopy images
Particle analysis: inclusion of a widefield and close-up electron microscopy image
density gradient
Separation method: density gradient, at least as validation of results attributed to EVs
EV density
Separation method: reporting of obtained EV density
ultracentrifugation specifics
Separation method: reporting of g-forces, duration and rotor type of ultracentrifugation steps
antibody specifics
Protein analysis: antibody clone/reference number and dilution
lysate preparation
Protein analysis: lysis buffer composition
Study data
Sample type
Cell culture supernatant
Sample origin
Deferiprone/iron chelation
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
DG + dUC
Protein markers
EV: HSP70/ Flotillin1
non-EV:
Proteomics
no
EV density (g/ml)
1.12-1.19 g/ml
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)/Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
Sample Condition
Deferiprone/iron chelation
EV-producing cells
C2C12
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 800 g and 10,000 g
Between 10,000 g and 50,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
Density gradient
Density medium
Sucrose
Type
Continuous
Lowest density fraction
0.4M
Highest density fraction
2.5M
Total gradient volume, incl. sample (mL)
12.4
Sample volume (mL)
0.2
Orientation
Bottom-up
Rotor type
SW 41 Ti
Speed (g)
188000
Duration (min)
16
Fraction volume (mL)
1 ml
Fraction processing
Centrifugation
Pelleting: volume per fraction
6 ml
Pelleting: duration (min)
65
Pelleting: rotor type
SW 32 Ti
Pelleting: speed (g)
100000
EV-subtype
Distinction between multiple subtypes
sedimentation speed;Other
Used subtypes
100,000g
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
Flotillin1/ HSP70
NA
EM
EM-type
Cryo-EM
Image type
Wide-field
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