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You searched for: EV230370 (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
EV230370 7/8 Sus scrofa Primary retinal pigmented epithelial cells DC
DG
(d)(U)C
UF 		Hernandez, Belinda J. 2023 75%

Study summary

Full title
Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (hide)
EV-METRIC
75% (96th 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
small 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
Density cushion
Density gradient
(Differential) (ultra)centrifugation
Ultrafiltration
Protein markers
EV: Syntenin-1/ ANXA2/ ITGB1
non-EV: Calreticulin/ Albumin/ Lamins/ Caspases
Proteomics
yes
EV density (g/ml)
1.07-1.11
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Sus scrofa
Sample Type
Cell culture supernatant
EV-producing cells
Primary retinal pigmented epithelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
99
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Speed (g)
200,000
Duration (min)
240
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: speed (g)
100,000
Density cushion
Density medium
Iodixanol
Sample volume
36
Cushion volume
2
Density of the cushion
60%
Centrifugation time
180
Centrifugation speed
100,000
Characterization: Protein analysis
Protein Concentration Method
Pierce 660 nm assay
Western Blot
Detected EV-associated proteins
Syntenin-1/ ANXA2/ ITGB1/ KRT10
Not detected contaminants
Calreticulin
Detected contaminants
Albumin
Not detected contaminants
Lamins/ Caspases
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
125.3
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.20E+06
EV230370 8/8 Sus scrofa Primary retinal pigmented epithelial cells DC
DG
(d)(U)C
UF 		Hernandez, Belinda J. 2023 75%

Study summary

Full title
Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (hide)
EV-METRIC
75% (96th 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
0.2mM H2O2
Focus vesicles
small 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
Density cushion
Density gradient
(Differential) (ultra)centrifugation
Ultrafiltration
Protein markers
EV: Syntenin-1/ ANXA2/ ITGB1
non-EV: Calreticulin/ Albumin/ Lamins/ Caspases
Proteomics
yes
EV density (g/ml)
1.07-1.11
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Sus scrofa
Sample Type
Cell culture supernatant
EV-producing cells
Primary retinal pigmented epithelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
99
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Speed (g)
200,000
Duration (min)
240
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: speed (g)
100,000
Density cushion
Density medium
Iodixanol
Sample volume
36
Cushion volume
2
Density of the cushion
60%
Centrifugation time
180
Centrifugation speed
100,000
Characterization: Protein analysis
Protein Concentration Method
Pierce 660 nm assay
Western Blot
Detected EV-associated proteins
Syntenin-1/ ANXA2/ ITGB1/ KRT10
Not detected contaminants
Calreticulin
Detected contaminants
Albumin
Not detected contaminants
Lamins/ Caspases
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
143.2
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 3.80E+06
EV230370 1/8 Homo sapiens iPSC-derived retinal pigmented epithelial cells DC
DG
(d)(U)C
UF 		Hernandez, Belinda J. 2023 50%

Study summary

Full title
Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (hide)
EV-METRIC
50% (87th 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
small 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
Density cushion
Density gradient
(Differential) (ultra)centrifugation
Ultrafiltration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
1.07-1.11
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
iPSC-derived retinal pigmented epithelial cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
99
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Speed (g)
200,000
Duration (min)
240
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: speed (g)
100,000
Density cushion
Density medium
Iodixanol
Sample volume
36
Cushion volume
2
Density of the cushion
60%
Centrifugation time
180
Centrifugation speed
100,000
Characterization: Protein analysis
Protein Concentration Method
Pierce 660 nm assay
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
Extra information
Proteomic data sets will be uploaded to publilcy available databases as aprtt of the publication process.
EV230370 2/8 Homo sapiens iPSC-derived retinal pigmented epithelial cells DC
DG
(d)(U)C
UF 		Hernandez, Belinda J. 2023 50%

Study summary

Full title
Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (hide)
EV-METRIC
50% (87th 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
Low genetic disease (Age-related macular degeneration) risk cell lines
Focus vesicles
small 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
Density cushion
Density gradient
(Differential) (ultra)centrifugation
Ultrafiltration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
1.07-1.11
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
iPSC-derived retinal pigmented epithelial cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
99
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Speed (g)
200,000
Duration (min)
240
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: speed (g)
100,000
Density cushion
Density medium
Iodixanol
Sample volume
36
Cushion volume
2
Density of the cushion
60%
Centrifugation time
180
Centrifugation speed
100,000
Characterization: Protein analysis
Protein Concentration Method
Pierce 660 nm assay
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230370 3/8 Homo sapiens iPSC-derived retinal pigmented epithelial cells DC
DG
(d)(U)C
UF 		Hernandez, Belinda J. 2023 50%

Study summary

Full title
Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (hide)
EV-METRIC
50% (87th 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
High genetic disease (Age-related macular degeneration) risk cell lines
Focus vesicles
small 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
Density cushion
Density gradient
(Differential) (ultra)centrifugation
Ultrafiltration
Protein markers
EV: None
non-EV: None
Proteomics
yes
EV density (g/ml)
1.07-1.11
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
iPSC-derived retinal pigmented epithelial cells
EV-harvesting Medium
Serum free medium
Cell viability (%)
99
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Pelleting performed
No
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
12
Sample volume (mL)
3
Orientation
Bottom-up
Speed (g)
200,000
Duration (min)
240
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: speed (g)
100,000
Density cushion
Density medium
Iodixanol
Sample volume
36
Cushion volume
2
Density of the cushion
60%
Centrifugation time
180
Centrifugation speed
100,000
Characterization: Protein analysis
Protein Concentration Method
Pierce 660 nm assay
Characterization: Lipid analysis
No
Characterization: Particle analysis
None
EV230370 4/8 Homo sapiens Primary fetal retinal pigmented epithelial cells (d)(U)C Hernandez, Belinda J. 2023 14%

Study summary

Full title
Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (hide)
EV-METRIC
14% (43rd 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
small 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
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Primary fetal retinal pigmented epithelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
99
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100,000
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Protein Yield (µg)
particles per milliliter of starting sample: 2.50E+06
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
125
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 2.50E+06
EV230370 5/8 Homo sapiens Primary fetal retinal pigmented epithelial cells (d)(U)C Hernandez, Belinda J. 2023 14%

Study summary

Full title
Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (hide)
EV-METRIC
14% (43rd 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
4 uM GW4869 treatment
Focus vesicles
small 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
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Primary fetal retinal pigmented epithelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
99
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100,000
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Protein Yield (µg)
particles per milliliter of starting sample: 1.20E+06
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
119.5
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 1.20E+06
EV230370 6/8 Homo sapiens Primary fetal retinal pigmented epithelial cells (d)(U)C Hernandez, Belinda J. 2023 14%

Study summary

Full title
Polarized desmosome and hemidesmosome shedding via small extracellular vesicles is an early indicator of outer blood-retina barrier dysfunction
All authors
Belinda J. Hernandez, Nikolai P. Skiba, Karolina Plössl, Madison Strain, Yutao Liu, Daniel Grigsby, Una Kelly, Martha A. Cady, Vikram Manocha, Arvydas Maminishkis, TeddiJo Watkins, Sheldon S. Miller, Allison Ashley-Koch, W. Daniel Stamer, Bernhard H. F. Weber, Catherine Bowes Rickman, Mikael Klingeborn
Journal
J Extracell Biol
Abstract
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photorec (show more...)The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of the eye, and is constantly exposed to oxidative stress. As such, dysfunction of the RPE underlies pathology leading to development of age-related macular degeneration (AMD), the leading cause of vision loss among the elderly in industrialized nations. A major responsibility of the RPE is to process photoreceptor outer segments, which relies on the proper functioning of its endocytic pathways and endosomal trafficking. Exosomes and other extracellular vesicles (EVs) from RPE are an essential part of these pathways and may be early indicators of cellular stress. To test the role of small EVs (sEVs) including exosomes, that may underlie the early stages of AMD, we used a polarized primary RPE cell culture model under chronic subtoxic oxidative stress. Unbiased proteomic analyses of highly purified basolateral sEVs from oxidatively stressed RPE cultures revealed changes in proteins involved in epithelial barrier integrity. There were also significant changes in proteins accumulating in the basal-side sub-RPE extracellular matrix during oxidative stress, that could be prevented with an inhibitor of sEV release. Thus, chronic subtoxic oxidative stress in primary RPE cultures induces changes in sEV content, including basal-side specific desmosome and hemidesmosome shedding via sEVs. These findings provide novel biomarkers of early cellular dysfunction and opportunity for therapeutic intervention in age-related retinal diseases (e.g., AMD). (hide)
EV-METRIC
14% (43rd 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
20 uM GW4869 treatment
Focus vesicles
small 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
Protein markers
EV: None
non-EV: None
Proteomics
no
Show all info
Study aim
Biogenesis/cargo sorting/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
Primary fetal retinal pigmented epithelial cells
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
>=18h at >= 100,000g
Cell viability (%)
99
Separation Method
(Differential) (ultra)centrifugation
dUC: 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
Pelleting performed
Yes
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100,000
Characterization: Protein analysis
None
Protein Concentration Method
Not determined
Protein Yield (µg)
particles per milliliter of starting sample: 6.00E+05
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
113.2
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 6.00E+05
1 - 8 of 8
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV230370
species
Sus
scrofa
Sus
scrofa
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
sample type
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
Cell
culture
cell type
Primary
retinal
pigmented
epithelial
cells
Primary
retinal
pigmented
epithelial
cells
iPSC-derived
retinal
pigmented
epithelial
cells
iPSC-derived
retinal
pigmented
epithelial
cells
iPSC-derived
retinal
pigmented
epithelial
cells
Primary
fetal
retinal
pigmented
epithelial
cells
Primary
fetal
retinal
pigmented
epithelial
cells
Primary
fetal
retinal
pigmented
epithelial
cells
medium
EV-depleted
medium
EV-depleted
medium
Serum
free
medium
Serum
free
medium
Serum
free
medium
EV-depleted
medium
EV-depleted
medium
EV-depleted
medium
condition
Control
condition
0.2mM
H2O2
Control
condition
Low
genetic
disease
(Age-related
macular
degeneration)
risk
cell
lines
High
genetic
disease
(Age-related
macular
degeneration)
risk
cell
lines
Control
condition
4
uM
GW4869
treatment
20
uM
GW4869
treatment
separation protocol
DC/
Density
gradient/
dUC/
Ultrafiltration
DC/
Density
gradient/
dUC/
Ultrafiltration
DC/
Density
gradient/
dUC/
Ultrafiltration
DC/
Density
gradient/
dUC/
Ultrafiltration
DC/
Density
gradient/
dUC/
Ultrafiltration
dUC
dUC
dUC
Exp. nr.
7
8
1
2
3
4
5
6
EV-METRIC %
75
75
50
50
50
14
14
14