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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV140335	1/1	Homo sapiens	NAY	(d)(U)C IAF	Gildea JJ	2014	14%
Study summary Full title Exosomal transfer from human renal proximal tubule cells to distal tubule and collecting duct cells All authors Gildea JJ, Seaton JE, Victor KG, Reyes CM, Bigler Wang D, Pettigrew AC, Courtner CE, Shah N, Tran HT, Van Sciver RE, Carlson JM, Felder RA Journal Clin Biochem Abstract OBJECTIVES: Exosomes are 50-90nm extracellular membrane particles that may mediate trans-cellular co (show more...)OBJECTIVES: Exosomes are 50-90nm extracellular membrane particles that may mediate trans-cellular communication between cells and tissues. We have reported that human urinary exosomes contain miRNA that are biomarkers for salt sensitivity and inverse salt sensitivity of blood pressure. This study examines exosomal transfer between cultured human renal proximal tubule cells (RPTCs) and from RPTCs to human distal tubule and collecting duct cells. DESIGN AND METHODS: For RPTC-to-RPTC exosomal transfer, we utilized 5 RPTC lines producing exosomes that were fluorescently labeled with exosomal-specific markers CD63-EGFP or CD9-RFP. Transfer between RPTCs was demonstrated by co-culturing CD63-EGFP and CD9-RFP stable clones and performing live confocal microscopy. For RPTC-to-distal segment exosomal transfer, we utilized 5 distal tubule and 3 collecting duct immortalized cell lines. RESULTS: Time-lapse videos revealed unique proximal tubule cellular uptake patterns for exosomes and eventual accumulation into the multivesicular body. Using culture supernatant containing exosomes from 3 CD9-RFP and 2 CD63-EGFP RPTC cell lines, all 5 distal tubule cell lines and all 3 collecting duct cell lines showed exosomal uptake as measured by microplate fluorometry. Furthermore, we found that RPTCs stimulated with fenoldopam (dopamine receptor agonist) had increased production of exosomes, which upon transfer to distal tubule and collecting duct cells, reduced the basal reactive oxygen species (ROS) production rates in those recipient cells. CONCLUSION: Due to the complex diversity of exosomal contents, this proximal-to-distal vesicular inter-nephron transfer may represent a previously unrecognized trans-renal communication system. (hide) EV-METRIC 14% (44th 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 IAF Protein markers EV: non-EV: Proteomics yes 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 10,000 g and 50,000 g Equal to or above 150,000 g Pelleting performed Yes Pelleting: time(min) 30 Immunoaffinity capture Selected surface protein(s) CD63 Characterization: Protein analysis

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EV-TRACK ID	EV140335
species	Homo sapiens
sample type	Cell culture
cell type	NAY
condition	NAY
separation protocol	(d)(U)C IAF
Exp. nr.	1
EV-METRIC %	14