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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
Details EV-TRACK ID Experiment nr. Species Sample type Isolation protocol First author Year EV-METRIC
EV130001 1/1 Homo sapiens Cell culture supernatant 0.2 µm filter
dUC
Yoshioka Y 2013 67%

Study summary

Full title
All authors
Yoshioka Y, Konishi Y, Kosaka N, Katsuda T, Kato T, Ochiya T
Journal
J Extracell Vesicles
Abstract
Several cell types, including tumour cells, secrete extracellular vesicles (EVs), and tumour-derived (show more...)Several cell types, including tumour cells, secrete extracellular vesicles (EVs), and tumour-derived EVs play a role in cancer initiation and progression. These vesicles include both a common set of membrane and cytosolic proteins and origin-specific subsets of proteins that likely correlated to cell type-associated functions. To confirm the presence of EVs in the preparations, researchers have identified so-called EV marker proteins, including the tetraspanin family proteins and such cytosolic proteins as heat shock 70 kDa protein 4 (HSP70) and tumour susceptibility gene 101 (TSG101). However, studies have shown that some EV markers are not always present in all EVs, which not only complicates the identification of EVs but also precludes the quantitative evaluation of EV proteins. Thus, it is strongly required to explore well-conserved EV marker proteins that are present at similar levels, regardless of their tissue or cellular origin. In this study, we compared the presence of 11 well-known EV marker proteins by immunoblotting using EVs isolated from 4 human prostate cell lines and 5 human breast cell lines, including cancer cells with different phenotypes. We found that all the tested EVs were positive for CD9 and CD81, with similar abundance that was irrespective of the EV origin. In contrast, other EV marker proteins, such as TSG101, Rab-5b and CD63, were detected in an inconsistent manner, depending on the origin of the EVs. Thus, we propose that the detection of CD9 and/or CD81 should ensure the presence of EVs. (hide)
EV-METRIC
67% (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
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
extracellular vesicles
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
0.2 µm filter + dUC
Protein markers
EV: CD63/ CD81/ CD9/ Flotilin1/ HSP70/ TSG101/ Caveolin1/ Rab5b/ Annexin2/ Integrin-beta1/ Actin
non-EV: Cell organelle protein
Proteomics
no
Show all info
Study aim
Biomarker
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
EV-harvesting Medium
serum free
Isolation Method
Differential ultra centrifugation
Differential UC: filtering steps
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
70
Wash: volume per pellet (ml)
11
Filtration steps
0.22µm or 0.2µm
Characterization: Protein analysis
Western Blot
Detected EV-associated proteins
CD63/ CD81/ CD9/ Flotilin1/ HSP70/ TSG101/ Caveolin1/ Rab5b/ Annexin2/ Integrin-beta1/ Actin
Detected contaminants
Cell organelle protein
ELISA
Detected EV-associated proteins
Caveolin1/ Rab5b/ Annexin2/ Integrin-beta1/ Actin
Characterization: Particle analysis
NTA
EM
EM-type
transmission EM
Image type
Close-up, Wide-field
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