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You searched for: EV180010 (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
Details EV-TRACK ID Experiment nr. Species Sample type Separation protocol First author Year EV-METRIC
EV180010 2/2 Homo sapiens Serum DG
(d)(U)C
Busatto, Sara 2018 88%

Study summary

Full title
All authors
Sara Busatto, Arianna Giacomini, Costanza Montis, Roberto Ronca, Paolo Bergese
Journal
Anal Chem
Abstract
Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capi (show more...)Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation, but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust, and versatile method for resolving dose-dependent uptake profiles of exosomes-the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications-by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed evaluation and comparison of the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, and A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth. (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. 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
Serum
Sample origin
Control condition
Focus vesicles
exosome
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
DG
(d)(U)C
Adj. k-factor
89.2 (pelleting)
Protein markers
EV: Alix/ CD81/ ANXA11/ CD63
non-EV: GM130/ APOA1
Proteomics
no
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Serum
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)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
89.20
Density gradient
Type
Discontinuous
Number of initial discontinuous layers
6
Lowest density fraction
0.15
Highest density fraction
0.6
Sample volume (mL)
0.8
Orientation
Bottom-up (sample migrates upwards)
Rotor type
MLS-50
Speed (g)
100000
Duration (min)
960
Fraction volume (mL)
0.4
Fraction processing
Centrifugation
Pelleting: volume per fraction
0.4
Pelleting: duration (min)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
89.20
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
Alix, CD63, CD81, ANXA11
Not detected contaminants
GM130, APOA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
DLS
Report type
Mean
Reported size (nm)
70
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
30-150
Other particle analysis name(2)
Colorimetric nanoplasmonic assay (CONAN)
EV-concentration
Yes
Particle yield
5.30E+12
EV180010 1/2 Homo sapiens Serum (d)(U)C
qEV
Busatto, Sara 2018 44%

Study summary

Full title
All authors
Sara Busatto, Arianna Giacomini, Costanza Montis, Roberto Ronca, Paolo Bergese
Journal
Anal Chem
Abstract
Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capi (show more...)Understanding extracellular vesicle (EV) internalization mechanisms and pathways in cells is of capital importance for both EV basic biology and clinical translation, but still presents analytical hurdles, such as undetermined purity grade and/or concentration of the EV samples and lack of standard protocols. We report an accessible, robust, and versatile method for resolving dose-dependent uptake profiles of exosomes-the nanosized (30-150 nm) subtypes of EVs of intracellular origin which are more intensively investigated for diagnostic and therapeutic applications-by cultured cells. The method is based on incubating recipient cells with consistently increasing doses of exosomes which are graded for purity and titrated by a COlorimetric NANoplasmonic (CONAN) assay followed by cell flow cytofluorimetric analysis. The proposed method allowed evaluation and comparison of the uptake of human serum exosomes by cancer cell lines of murine (TRAMP-C2) and human (LNCaP, DU145, MDA-MB-231, and A375) origin, setting a firmer footing for better characterization and understanding of exosome biology in different in vitro and (potentially) in vivo models of cancer growth. (hide)
EV-METRIC
44% (86th 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
Serum
Sample origin
Control condition
Focus vesicles
exosome
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
qEV
Adj. k-factor
89.2 (pelleting)
Protein markers
EV: ANXA11/ CD63
non-EV: GM130/ APOA1
Proteomics
no
Show all info
Study aim
Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Serum
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)
120
Pelleting: rotor type
TLA-55
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
89.20
Commercial kit
qEV
Other
Name other separation method
qEV
Characterization: Protein analysis
Protein Concentration Method
Not determined
Western Blot
Detected EV-associated proteins
CD63, ANXA11
Not detected contaminants
GM130, APOA1
Characterization: Lipid analysis
No
Characterization: Particle analysis
EM
EM-type
Atomic force-EM
Image type
Wide-field
Report size (nm)
30-150
1 - 2 of 2
  • CM = Commercial method
  • dUC = differential ultracentrifugation
  • DG = density gradient
  • UF = ultrafiltration
  • SEC = size-exclusion chromatography
EV-TRACK ID
EV180010
species
Homo sapiens
sample type
Serum
condition
Control condition
separation protocol
DG
(d)(U)C
(d)(U)C
qEV
Exp. nr.
2
1
EV-METRIC %
88
44