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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
EV160001 1/1 Mus musculus Cell culture supernatant DG
dUC
Stremersch S 2016 87%

Study summary

Full title
All authors
Stremersch S, Vandenbroucke RE, Van Wonterghem E, Hendrix A, De Smedt SC, Raemdonck K
Journal
J Control Release
Abstract
Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natu (show more...)Exosome-like vesicles (ELVs) play an important role in intercellular communication by acting as natural carriers for biomolecule transfer between cells. This unique feature rationalizes their exploitation as bio-inspired drug delivery systems. However, the therapeutic application of ELVs is hampered by the lack of efficient and reproducible drug loading methods, in particular for therapeutic macromolecules. To overcome this limitation, we present a generic method to attach siRNA to the surface of isolated ELVs by means of a cholesterol anchor. Despite a feasible uptake in both a dendritic and lung epithelial cell line, B16F10- and JAWSII-derived ELVs were unable to functionally deliver the associated small RNAs, neither exogenous cholesterol-conjugated siRNA nor endogenous miRNA derived from the melanoma producer cell. The latter results were confirmed both for purified ELVs and ELVs delivered via a transwell co-culture set-up. In contrast, simple anionic fusogenic liposomes were able to induce a marked siRNA-mediated gene knockdown under equal experimental conditions, both indicating successful cytosolic delivery of surface-bound cholesterol-conjugated siRNA and further underscoring the incapacity of the here evaluated ELVs to guide cytosolic delivery of small RNAs. In conclusion, we demonstrate that a more in-depth understanding of the biomolecular delivery mechanism and specificity is required before ELVs can be envisioned as a generic siRNA carrier. (hide)
EV-METRIC
87% (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-like 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
DG + dUC
Protein markers
EV: CD63/ CD81/ HSP70/ beta-actin
non-EV: Calreticulin/ GM130
Proteomics
no
Show all info
Study aim
Function, Mechanism of uptake/transfer
Sample
Species
Mus musculus
Sample Type
Cell culture supernatant
EV-producing cells
B16F10, JAWSII
EV-harvesting Medium
EV-depleted serum
Origin
Control condition
Preparation of EDS
Ultrafiltration (MWCO 300 kDa)
Cell viability (%)
95
Isolation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 10,000 g and 50,000 g
Density gradient
Only used for validation of main results
Yes
Density medium
115.5
Type
Discontinuous
Number of initial discontinuous layers
5
Lowest density fraction
0.125
Highest density fraction
0.5
Sample volume (mL)
1
Orientation
Top-down (sample migrates downwards)
Rotor type
SW 55 Ti
Speed (g)
200000
Duration (min)
900
Fraction volume (mL)
1
Fraction processing
Centrifugation
Pelleting: volume per fraction
5
Pelleting: duration (min)
150
Pelleting: rotor type
SW 55 Ti
Pelleting: speed (g)
120000
Pelleting: adjusted k-factor
115.5
Pelleting-wash: volume per pellet (mL)
5
Pelleting-wash: duration (min)
70
Pelleting-wash: rotor type
115.5
Pelleting-wash: speed (g)
SW 55 Ti
Pelleting-wash: adjusted k-factor
115.5
EV-subtype
Used subtypes
NO
Characterization: Protein analysis
Protein Concentration Method
BCA
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
CD63, CD81, HSP70, beta-actin
Flow cytometry specific beads
Selected surface protein(s)
CD63
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
30-300
EV concentration
Yes
Particle yield
1000
EM
EM-type
Cryo-EM
Image type
Close-up
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