EV-TRACK

Search > Results

You searched for: EV190008 (EV-TRACK ID)

Showing 1 - 2 of 2

Results list Study Tree

Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV190008	2/2	Homo sapiens	HEK 293	(d)(U)C ExoQuick Filtration	Duong N	2019	38%
Study summary Full title Decoy exosomes as a novel biologic reagent to antagonize inflammation. All authors Duong N, Curley K, Brown A, Campanelli A, Do MA, Levy D, Tantry A, Marriott G, Lu B. Journal Nanomedicine Abstract Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to (show more...)Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα. (hide) EV-METRIC 38% (79th 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 TNFalpha-CD63-GFP overexpressing 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 ExoQuick Filtration Protein markers EV: / TSG101/ CD63/ CD81/ Alix/ ICAM/ Flotillin1/ HSP70 non-EV: / GM130 Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK 293 EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Protein Concentration Method Other;UV-Vis Microvolume Spectrophotometer Western Blot Antibody details provided? No Detected EV-associated proteins Not detected contaminants ELISA Antibody details provided? No Detected EV-associated proteins Flow cytometry Type of Flow cytometry Calibration bead size Antibody details provided? No Detected EV-associated proteins Other 1 Dot blot Detected EV-associated proteins Flotillin1/ Alix/ CD63/ ICAM/ TSG101/ HSP70/ CD81 Not detected contaminants GM130 Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 46

	EV190008	1/2	Homo sapiens	HEK 293	(d)(U)C ExoQuick Filtration	Duong N	2019	0%
Study summary Full title Decoy exosomes as a novel biologic reagent to antagonize inflammation. All authors Duong N, Curley K, Brown A, Campanelli A, Do MA, Levy D, Tantry A, Marriott G, Lu B. Journal Nanomedicine Abstract Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to (show more...)Background: Exosomes are ubiquitous naturally secreted stable nanovesicles that can be engineered to target and deliver novel therapeutics to treat a host of human diseases. Methods: We engineered the surfaces of cell-derived nanovesicles to act as decoys in the treatment of inflammation by antagonizing the major proinflammatory cytokine, tumor necrosis factor alpha (TNFα). Results: Decoy exosomes were generated by displaying the TNFα binding domain of human TNF receptor-1 (hTNFR1) on the outer surface of exosomes using stably transfected HEK293 cells. We developed an efficient method to purify the engineered exosomes from conditioned medium based on sequential centrifugation, ultrafiltration, and precipitation. We characterized decoy exosomes using immune-quantification, nanoparticle tracking analysis, and confocal microscopy to confirm that they retain the correct orientation, size, and shape of naturally produced exosomes. We demonstrated the engineered decoy exosomes specifically antagonize activities of TNFα using an inflammatory reporter cell line. Conclusions: Decoy exosomes produced in human cells serve as a novel biologic reagent for antagonizing inflammatory signaling mediated by TNFα. (hide) EV-METRIC 0% (median: 14% 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 RFP-CD63-GFP overexpressing 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 ExoQuick Filtration Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells HEK 293 EV-harvesting Medium Serum free medium Cell viability (%) 85 Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 800 g and 10,000 g Pelleting performed No Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis None Protein Concentration Method Other;UV-Vis Microvolume Spectrophotometer Western Blot Antibody details provided? No Detected EV-associated proteins Not detected contaminants ELISA Antibody details provided? No Detected EV-associated proteins Flow cytometry Type of Flow cytometry Calibration bead size Antibody details provided? No Detected EV-associated proteins Characterization: Lipid analysis No Characterization: Particle analysis NTA Report type Modus Reported size (nm) 48

				1 - 2 of 2

EV-TRACK ID	EV190008
species	Homo sapiens
sample type	Cell culture
cell type	HEK 293
condition	TNFalpha-CD63-GFP overexpressing	RFP-CD63-GFP overexpressing
separation protocol	(d)(U)C ExoQuick Filtration	(d)(U)C ExoQuick Filtration
Exp. nr.	2	1
EV-METRIC %	38	0