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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV180011	1/1	Homo sapiens	HEK293	DG (d)(U)C Filtration	Kathrin Gärtner	2019	100%
Study summary Full title Engineering extracellular vesicles as novel treatment options: exploiting herpesviral immunity in CLL 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. 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 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) (ultra)centrifugation DG = density gradient UF = ultrafiltration SEC = size-exclusion chromatography IAF = immuno-affinity capture DG (d)(U)C 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 EV-producing cells HEK293 EV-harvesting Medium EV-depleted serum Preparation of EDS overnight (16h) at >=100,000g Cell viability (%) NA Separation Method (Differential) (ultra)centrifugation 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 performed Yes Pelleting: time(min) 120 Pelleting: rotor type SW 28 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 253.9 Density gradient 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 Detected EV-associated proteins Alix, CD63, TSG101 Not detected contaminants Calnexin Characterization: RNA analysis Database No Proteinase treatment No RNAse treatment Yes Moment of RNAse treatment After RNAse type RNase A RNAse concentration 0.01 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Size range/distribution Reported size (nm) 50-300 EV concentration Yes EM EM-type Transmission-EM/ Immune-EM EM protein CD63 Image type Close-up, Wide-field

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EV-TRACK ID	EV180011
species	Homo sapiens
sample type	Cell culture
cell type	HEK293
condition	Overexpressing gp350 CD40L and/or pp65
separation protocol	DG (d)(U)C Filtration
Exp. nr.	1
EV-METRIC %	100