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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170063	1/2	Homo sapiens	Jurkat	DG (d)(U)C Filtration	Nemeth, Andrea	2017	75%
Study summary Full title Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA. All authors Németh A, Orgovan N, Sódar BW, Osteikoetxea X, Pálóczi K, Szabó-Taylor KÉ, Vukman KV, Kittel Á, Turiák L, Wiener Z, Tóth S, Drahos L, Vékey K, Horvath R, Buzás EI. Journal Sci Rep Abstract Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesi (show more...)Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes. (hide) EV-METRIC 75% (96th 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 Cyprofloxacin 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 DG (d)(U)C Filtration Adj. k-factor 115.3 (pelleting) / 115.3 (washing) Protein markers EV: CD63/ HistoneH2B non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type MLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 115.3 Wash: time (min) 70 Wash: Rotor Type MLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 115.3 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 180 Pelleting: speed (g) 100000 Filtration steps > 0.45 µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

	EV170063	2/2	Homo sapiens	Jurkat	DG (d)(U)C Filtration	Nemeth, Andrea	2017	75%
Study summary Full title Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA. All authors Németh A, Orgovan N, Sódar BW, Osteikoetxea X, Pálóczi K, Szabó-Taylor KÉ, Vukman KV, Kittel Á, Turiák L, Wiener Z, Tóth S, Drahos L, Vékey K, Horvath R, Buzás EI. Journal Sci Rep Abstract Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesi (show more...)Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes. (hide) EV-METRIC 75% (96th 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 Control condition 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 DG (d)(U)C Filtration Adj. k-factor 115.3 (pelleting) / 115.3 (washing) Protein markers EV: CD63 non-EV: None Proteomics yes Show all info Study aim Biogenesis/cargo sorting, Identification of content (omics approaches) Sample Species Homo sapiens Sample Type Cell culture supernatant EV-producing cells Jurkat EV-harvesting Medium Serum free medium Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Below or equal to 800 g Between 800 g and 10,000 g Between 10,000 g and 50,000 g Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 70 Pelleting: rotor type MLA-55 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 115.3 Wash: time (min) 70 Wash: Rotor Type MLA-55 Wash: speed (g) 100000 Wash: adjusted k-factor 115.3 Density gradient Only used for validation of main results Yes Type Discontinuous Number of initial discontinuous layers 4 Lowest density fraction 0.05 Highest density fraction 0.4 Sample volume (mL) 0.5 Orientation Top-down (sample migrates downwards) Rotor type MLS-50 Speed (g) 100000 Duration (min) 1080 Fraction volume (mL) 0.5 Fraction processing Centrifugation Pelleting: duration (min) 180 Pelleting: speed (g) 100000 Filtration steps > 0.45 µm, 0.22µm or 0.2µm Characterization: Protein analysis Protein Concentration Method Not determined Proteomics database No Characterization: Lipid analysis Yes Characterization: Particle analysis TRPS Report type Size range/distribution Reported size (nm) 50-200 EV concentration Yes EM EM-type Transmission-EM Image type Wide-field

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EV-TRACK ID	EV170063
species	Homo sapiens
sample type	Cell culture
cell type	Jurkat
condition	Cyprofloxacin	Control condition
separation protocol	DG (d)(U)C Filtration	DG (d)(U)C Filtration
Exp. nr.	1	2
EV-METRIC %	75	75