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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.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
Details EV-TRACK ID Experiment nr. Species Sample type separation protocol First author Year EV-METRIC
EV180011 1/1 Homo sapiens Cell culture supernatant DG
dUC
Filtration
Kathrin Gärtner 2019 100%

Study summary

Full title
All authors
Kathrin Gärtner, Manja Luckner, Gerhard Wanner, Reinhard Zeidler
Journal
J Extracell Vesicles
Abstract
Extracellular vesicles (EVs) are important mediators of cell–cell communication. Intriguingly, EVs (show more...)Extracellular vesicles (EVs) are important mediators of cell–cell communication. Intriguingly, EVs can be engineered and thus exploited for the targeted transfer of functional proteins of interest. Thus, engineered EVs may constitute attractive tools for the development of novel therapeutic interventions, like cancer immunotherapies, vaccinations or targeted drug delivery. Here, we describe a novel experimental immunotherapeutic approach for the adjuvant treatment of chronic lymphocytic leukaemia (CLL) based on engineered EVs carrying gp350, the major glycoprotein of Epstein–Barr virus (EBV), CD40L, a central immune accessory molecule and pp65, an immunodominant antigen of the human cytomegalovirus (CMV). We show that these engineered EVs specifically interact with malignant B cells from CLL patients and render these cells immunogenic to allogeneic and autologous EBV- and CMV-specific CD4+ and CD8+ T cells. Collectively, co-opting engineered EVs to re-target the strong herpesviral immunity in CLL patients to malignant cells constitutes an attractive strategy for the adjuvant treatment of a still incurable disease. (hide)
EV-METRIC
100% (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
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
Overexpressing gp350, CD40L and/or pp65
Focus vesicles
extracellular vesicle
Separation protocol
Separation protocol
  • Gives a short, non-chronological overview of the
    different steps of the separation protocol.
    • dUC = differential ultracentrifugation
    • DG = density gradient
    • UF = ultrafiltration
    • SEC = size-exclusion chromatography
DG + dUC + Filtration
Adj. k-factor
253.9 (pelleting)
Protein markers
EV: Alix/ TSG101/ CD63
non-EV: Calnexin
Proteomics
no
Show all info
Study aim
Function, Mechanism of uptake/transfer
Sample
Species
Homo sapiens
Sample Type
Cell culture supernatant
Sample Condition
Overexpressing gp350, CD40L and/or pp65
EV-producing cells
HEK293
EV-harvesting Medium
EV-depleted serum
Preparation of EDS
overnight (16h) at >=100,000g
Cell viability
95
Separation Method
Differential ultracentrifugation
dUC: centrifugation steps
Below or equal to 800 g
Between 800 g and 10,000 g
Between 100,000 g and 150,000 g
Pelleting: time(min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Density gradient
Density medium
Iodixanol
Type
Discontinuous
Number of initial discontinuous layers
2
Lowest density fraction
0.3
Highest density fraction
0.44
Sample volume (mL)
0.5
Orientation
Bottom-up (sample migrates upwards)
Rotor type
SW 60 Ti
Speed (g)
160000
Duration (min)
960
Fraction volume (mL)
0.5
Fraction processing
Centrifugation
Pelleting: volume per fraction
30
Pelleting: duration (min)
120
Pelleting: rotor type
SW 28
Pelleting: speed (g)
100000
Pelleting: adjusted k-factor
253.9
Characterization: Protein analysis
Protein Concentration Method
Bradford
Western Blot
Antibody details provided?
Yes
Lysis buffer provided?
Yes
Detected EV-associated proteins
Alix, CD63, TSG101
Not detected contaminants
Calnexin
Characterization: RNA analysis
RNAse treatment
Moment of RNAse treatment
After
RNAse type
RNase A
RNAse concentration
0.01
Characterization: Particle analysis
NTA
Report type
Size range/distribution
Reported size (nm)
50-300
EV concentration
Yes
EM
EM-type
Transmission-EM/ Immune-EM
Proteïns
CD63
Image type
Close-up, Wide-field
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