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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140275	1/2	Homo sapiens	NAY	(d)(U)C Filtration	Jella KK	2014	29%
Study summary Full title Exosomes are involved in mediating radiation induced bystander signaling in human keratinocyte cells All authors Jella KK, Rani S, O'Driscoll L, McClean B, Byrne HJ, Lyng FM Journal Radiat Res Abstract There is much evidence supporting the existence of bystander effects in cells that were never expose (show more...)There is much evidence supporting the existence of bystander effects in cells that were never exposed to radiation. Directly irradiated cells and bystander cells can communicate with each other using gap junctional intercellular communication or by releasing soluble factors into the surrounding medium. Exosomes and microvesicles are also known to mediate communication between cells. The main aim of this study is to establish whether exosomes and microvesicles are involved in radiation induced bystander signaling. Human keratinocytes, HaCaT cells, were irradiated (0.005, 0.05 and 0.5 Gy) using ? rays produced from a cobalt 60 teletherapy unit. After irradiation, the cells were incubated for 1 h and the irradiated cell conditioned medium (ICCM) was harvested. Exosomes were isolated from the ICCM using ultracentrifugation. Exosomes were characterized using light scattering analysis (LSA) and scanning transmission electron microscopy (STEM). Cytotoxicity and reactive oxygen species assays and real time calcium imaging were performed either with ICCM from which exosomes and microvesicles were removed or with the exosome fraction resuspended in cell culture media. The characterization data showed a particle size distribution indicative of both exosomes (30-100 nm) and microvesicles (>100 nm) and the light scattering analysis showed increased concentration of both exosomes and microvesicles with increasing dose. Western blotting confirmed the presence of an exosomal protein marker, TSG 101. Treatment of unirradiated cells with ICCM in which exosomes and microvesicles were removed resulted in abrogation of ICCM induced effects such as reduction in viability, calcium influx and production of reactive oxygen species. Addition of exosomes to fresh media produced similar effects to complete ICCM. These results suggest a role for exosomes and microvesicles in radiation induced bystander signaling. (hide) EV-METRIC 29% (68th 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 NAY Focus vesicles exosomes 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 Filtration Adj. k-factor 126.8 (pelleting) / 126.8 (washing) Protein markers EV: non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method (Differential) (ultra)centrifugation dUC: centrifugation steps Between 100,000 g and 150,000 g Pelleting performed Yes Pelleting: time(min) 60 Pelleting: rotor type 80Ti Pelleting: adjusted k-factor 126.8 Wash: Rotor Type 80Ti Wash: adjusted k-factor 126.8 Filtration steps 0.22µm or 0.2µm Characterization: Particle analysis NTA EM EM-type transmission EM Image type Wide-field

	EV140275	2/2	Homo sapiens	NAY	ExoQuick Filtration	Jella KK	2014	0%
Study summary Full title Exosomes are involved in mediating radiation induced bystander signaling in human keratinocyte cells All authors Jella KK, Rani S, O'Driscoll L, McClean B, Byrne HJ, Lyng FM Journal Radiat Res Abstract There is much evidence supporting the existence of bystander effects in cells that were never expose (show more...)There is much evidence supporting the existence of bystander effects in cells that were never exposed to radiation. Directly irradiated cells and bystander cells can communicate with each other using gap junctional intercellular communication or by releasing soluble factors into the surrounding medium. Exosomes and microvesicles are also known to mediate communication between cells. The main aim of this study is to establish whether exosomes and microvesicles are involved in radiation induced bystander signaling. Human keratinocytes, HaCaT cells, were irradiated (0.005, 0.05 and 0.5 Gy) using ? rays produced from a cobalt 60 teletherapy unit. After irradiation, the cells were incubated for 1 h and the irradiated cell conditioned medium (ICCM) was harvested. Exosomes were isolated from the ICCM using ultracentrifugation. Exosomes were characterized using light scattering analysis (LSA) and scanning transmission electron microscopy (STEM). Cytotoxicity and reactive oxygen species assays and real time calcium imaging were performed either with ICCM from which exosomes and microvesicles were removed or with the exosome fraction resuspended in cell culture media. The characterization data showed a particle size distribution indicative of both exosomes (30-100 nm) and microvesicles (>100 nm) and the light scattering analysis showed increased concentration of both exosomes and microvesicles with increasing dose. Western blotting confirmed the presence of an exosomal protein marker, TSG 101. Treatment of unirradiated cells with ICCM in which exosomes and microvesicles were removed resulted in abrogation of ICCM induced effects such as reduction in viability, calcium influx and production of reactive oxygen species. Addition of exosomes to fresh media produced similar effects to complete ICCM. These results suggest a role for exosomes and microvesicles in radiation induced bystander signaling. (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 NAY Focus vesicles exosomes 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 ExoQuick Filtration Protein markers EV: TSG101 non-EV: Proteomics no Show all info Study aim Function Sample Species Homo sapiens Sample Type Cell culture supernatant EV-harvesting Medium serum free Separation Method Filtration steps 0.22µm or 0.2µm Commercial kit ExoQuick Other Name other separation method ExoQuick Characterization: Protein analysis Western Blot Antibody details provided? No Detected EV-associated proteins TSG101 Characterization: Particle analysis None

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EV-TRACK ID	EV140275
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C Filtration	ExoQuick Filtration
Exp. nr.	1	2
EV-METRIC %	29	0