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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV170017	1/1	Mus musculus	Bronchoalveolar lavage fluid	(d)(U)C	Maroto, Rosario	2017	75%
Study summary Full title Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses. All authors Maroto R, Zhao Y, Jamaluddin M, Popov VL, Wang H, Kalubowilage M, Zhang Y, Luisi J, Sun H, Culbertson CT, Bossmann SH, Motamedi M, Brasier AR Journal J Extracell Vesicles Abstract Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and (show more...)Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or -80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at -80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at -80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface characteristics, morphological features and protein content of BALF exosomes. For preservation of the exosome protein content and representative functional analysis, airway exosomes should be analysed immediately after isolation. (hide) EV-METRIC 75% (92nd 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 Bronchoalveolar lavage fluid Sample origin poly IC stimulated 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 Adj. k-factor 60.38 (pelleting) / 60.38 (washing) Protein markers EV: Alix/ CD63,HSP90,Alix/ HSP90/ CD63 non-EV: Grp94,beta-actin/ Grp94/ beta-actin Proteomics yes Show all info Study aim Identification of content (omics approaches) Sample Species Mus musculus Sample Type Bronchoalveolar lavage fluid 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 TLA-100.3 Pelleting: speed (g) 100000 Pelleting: adjusted k-factor 60.38 Wash: time (min) 60 Wash: Rotor Type TLA-100.3 Wash: speed (g) 100000 Wash: adjusted k-factor 60.38 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD63,HSP90,Alix Not detected contaminants Grp94,beta-actin Proteomics database No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Median Reported size (nm) 95 EM EM-type Transmission-EM Image type Close-up, Wide-field

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EV-TRACK ID	EV170017
species	Mus musculus
sample type	Bronchoalveolar lavage fluid
condition	poly IC stimulated
separation protocol	(d)(U)C
Exp. nr.	1
EV-METRIC %	75