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You searched for: EV210262 (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
Details EV-TRACK code Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV210262 1/2 Homo sapiens Cell culture supernatant (d)(U)C
DG
Filtration
Flores-Bellver, Miguel 2021 89%

Study summary

Full title
All authors
Miguel Flores-Bellver, Jason Mighty, Silvia Aparicio-Domingo, Kang V Li, Cui Shi, Jing Zhou, Hannah Cobb, Patrick McGrath, German Michelis, Patricia Lenhart, Ganna Bilousova, Søren Heissel, Michael J Rudy, Christina Coughlan 10 , Andrew E Goodspeed 11 12 , S Patricia Becerra, Stephen Redenti 13 , M Valeria Canto-Soler
Journal
J Extracell Vesicles
Abstract
Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key con (show more...)Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth. (hide)
EV-METRIC
89% (98th 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
Density gradient
Filtration
Adj. k-factor
7.66 (washing)
Protein markers
EV: CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101
non-EV: GM130
Proteomics
yes
EV density (g/ml)
1.064
Show all info
Study aim
Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hiPSC-derived RPE
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability (%)
99
Cell count
8400000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 70.1Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
10
Wash: time (min)
90
Wash: Rotor Type
Type 70.1Ti
Wash: speed (g)
120000
Wash: adjusted k-factor
7.66E
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
10
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
141000
Duration (min)
3600
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
9
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
7.66E
Filtration steps
0.2 or 0.22 ?m
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
15
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101
Detected contaminants
GM130
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
120
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 4.00e+10
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
120
EV210262 2/2 Homo sapiens Cell culture supernatant (d)(U)C
DG
Filtration
Flores-Bellver, Miguel 2021 89%

Study summary

Full title
All authors
Miguel Flores-Bellver, Jason Mighty, Silvia Aparicio-Domingo, Kang V Li, Cui Shi, Jing Zhou, Hannah Cobb, Patrick McGrath, German Michelis, Patricia Lenhart, Ganna Bilousova, Søren Heissel, Michael J Rudy, Christina Coughlan 10 , Andrew E Goodspeed 11 12 , S Patricia Becerra, Stephen Redenti 13 , M Valeria Canto-Soler
Journal
J Extracell Vesicles
Abstract
Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key con (show more...)Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth. (hide)
EV-METRIC
89% (98th 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
Cigarette smoke treatment
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
Density gradient
Filtration
Adj. k-factor
7.66 (washing)
Protein markers
EV: CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101
non-EV: GM130
Proteomics
yes
EV density (g/ml)
1.064
Show all info
Study aim
Mechanism of uptake/transfer/Identification of content (omics approaches)
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-producing cells
hiPSC-derived RPE
EV-harvesting Medium
EV-depleted medium
Preparation of EDS
Commercial EDS
Cell viability (%)
99
Cell count
8400000
Separation Method
(Differential) (ultra)centrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting performed
Yes
Pelleting: time(min)
90
Pelleting: rotor type
Type 70.1Ti
Pelleting: speed (g)
120000
Wash: volume per pellet (ml)
10
Wash: time (min)
90
Wash: Rotor Type
Type 70.1Ti
Wash: speed (g)
120000
Wash: adjusted k-factor
7.66E
Density gradient
Only used for validation of main results
Yes
Type
Continuous
Lowest density fraction
5%
Highest density fraction
40%
Total gradient volume, incl. sample (mL)
10
Sample volume (mL)
1
Orientation
Top-down
Rotor type
SW 41 Ti
Speed (g)
141000
Duration (min)
3600
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
9
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
7.66E
Filtration steps
0.2 or 0.22 ?m
Characterization: Protein analysis
Protein Concentration Method
microBCA
Protein Yield (µg)
15
Western Blot
Antibody details provided?
Yes
Antibody dilution provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63/ Flotillin-1/ HSP70/ HSP90/ TSG101
Detected contaminants
GM130
Proteomics database
Yes
Characterization: Lipid analysis
No
Characterization: Particle analysis
NTA
Report type
Mean
Reported size (nm)
120
EV concentration
Yes
Particle yield
particles per milliliter of starting sample: 4.70e+11
EM
EM-type
Transmission-EM
Image type
Close-up
Report size (nm)
120
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV210262
species
Homo sapiens
sample type
Cell culture
cell type
hiPSC-derived RPE
condition
Control condition
Cigarette
smoke treatment
separation protocol
dUC/
Density gradient/ Filtration
dUC/
Density gradient/ Filtration
Exp. nr.
1
2
EV-METRIC %
89
89