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You searched for: EV170046 (EV-TRACK ID)

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EV-TRACK ID
species
sample_type
EV_cell_name
medium
subject_condition
isolation_protocols
EV_subtype_diff
vesilerelated_term
case_nr
EV_metric_score
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV170046
species
Sus scrofa
sample type
Cell culture
cell type
Primary
retinal pigmented
epithelium cells
condition
Basolateral
side of polarized layer
Apical
side of polarized layer
isolation protocol
DG
dUC
Ultrafiltration
DG
dUC
Ultrafiltration
case number
1
2
EV-METRIC %
88
88
Experiment number
  • If needed, multiple experiments were identified in a single publication based on differing sample types, isolation protocols and/or vesicle types of interest.
Species
  • Species of origin of the EVs.
Isolation protocol
  • Gives a short, non-chronological overview of the different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Experiment number
  • Experiments differ in Vesicle type
Experiment number
  • Experiments differ in Vesicle type
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV170046 1/2 Sus scrofa Cell culture supernatant DG
dUC
Ultrafiltration
Klingeborn M 2017 88%

Study summary

Full title
All authors
Klingeborn M, Dismuke WM, Skiba NP, Kelly U, Stamer WD, Bowes Rickman C
Journal
Sci Rep
Abstract
The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its pola (show more...)The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers. (hide)
EV-METRIC
88% (99th 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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Ultrafiltration
Adj. k-factor
253.9 (pelleting) / 253.9 (washing)
Protein markers
EV: TSG101/ Syntenin-1
non-EV: Calreticulin
Proteomics
yes
Show all info
Study aim
New methodological development, Identification of content (omics approaches)
Sample
Species
Sus scrofa
Sample Type
Cell culture supernatant
Sample Condition
Basolateral side of polarized layer
EV-producing cells
Primary retinal pigmented epithelium cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Wash: time (min)
90
Wash: Rotor Type
SW 28
Wash: speed (g)
100000
Wash: adjusted k-factor
253.9
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
1
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
90
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Characterization: Protein analysis
Protein Concentration Method
Pierce 660nm Protein Assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
TSG101, Syntenin-1
Not detected contaminants
Calreticulin
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
132.2±13.1
EM
EM-type
Transmission-EM
Image type
Wide-field
EV170046 2/2 Sus scrofa Cell culture supernatant DG
dUC
Ultrafiltration
Klingeborn M 2017 88%

Study summary

Full title
All authors
Klingeborn M, Dismuke WM, Skiba NP, Kelly U, Stamer WD, Bowes Rickman C
Journal
Sci Rep
Abstract
The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its pola (show more...)The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in the eye and its polarity is responsible for directional secretion and uptake of proteins, lipoprotein particles and extracellular vesicles (EVs). Such a secretional division dictates directed interactions between the systemic circulation (basolateral) and the retina (apical). Our goal is to define the polarized proteomes and physical characteristics of EVs released from the RPE. Primary cultures of porcine RPE cells were differentiated into polarized RPE monolayers on permeable supports. EVs were isolated from media bathing either apical or basolateral RPE surfaces, and two subpopulations of small EVs including exosomes, and dense EVs, were purified and processed for proteomic profiling. In parallel, EV size distribution and concentration were determined. Using protein correlation profiling mass spectrometry, a total of 631 proteins were identified in exosome preparations, 299 of which were uniquely released apically, and 94 uniquely released basolaterally. Selected proteins were validated by Western blot. The proteomes of these exosome and dense EVs preparations suggest that epithelial polarity impacts directional release. These data serve as a foundation for comparative studies aimed at elucidating the role of exosomes in the molecular pathophysiology of retinal diseases and help identify potential therapeutic targets and biomarkers. (hide)
EV-METRIC
88% (99th 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
Isolation method: density gradient, at least as validation of results attributed to EVs
EV density
Isolation method: reporting of obtained EV density
ultracentrifugation specifics
Isolation 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
Focus vesicles
exosome
Isolation protocol
Isolation protocol
  • Gives a short, non-chronological overview of the
    different steps of the isolation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Ultrafiltration
Adj. k-factor
253.9 (pelleting) / 253.9 (washing)
Protein markers
EV: TSG101/ Syntenin-1
non-EV: Calreticulin
Proteomics
yes
Show all info
Study aim
New methodological development, Identification of content (omics approaches)
Sample
Species
Sus scrofa
Sample Type
Cell culture supernatant
Sample Condition
Apical side of polarized layer
EV-producing cells
Primary retinal pigmented epithelium cells
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
90
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Wash: time (min)
90
Wash: Rotor Type
SW 28
Wash: speed (g)
100000
Wash: adjusted k-factor
253.9
Density gradient
Only used for validation of main results
Yes
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
4
Lowest density fraction
0.05
Highest density fraction
0.4
Sample volume (mL)
1
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 41 Ti
Speed (g)
100000
Duration (min)
1080
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
12
Pelleting: duration (min)
90
Pelleting: rotor type
SW 41 Ti
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
255.8
Ultra filtration
Cut-off size (kDa)
100
Membrane type
Polyethersulfone (PES)
Characterization: Protein analysis
Protein Concentration Method
Pierce 660nm Protein Assay
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
TSG101, Syntenin-1
Not detected contaminants
Calreticulin
Proteomics database
No
Characterization: Particle analysis
NTA
Report type
Modus
Reported size (nm)
118.6±9.9
EM
EM-type
Transmission-EM
Image type
Close-up
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