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Details	EV-TRACK ID	Experiment nr.	Species	Sample type	Separation protocol	First author	Year	EV-METRIC
	EV200078	1/1	Homo sapiens	saliva	SEC (non-commercial) Filtration UF	Ogawa, Yuko	2021	75%
Study summary Full title Stability of human salivary extracellular vesicles containing dipeptidyl peptidase IV under simulated gastrointestinal tract conditions All authors Yuko Ogawa, Yoshihiro Akimoto, Mamoru Ikemoto, Yoshikuni Goto, Anna Ishikawa, Sakura Ohta, Yumi Takase, Hayato Kawakami, Masafumi Tsujimoto, Ryohei Yanoshita Journal Biochemistry and Biophysics Reports Abstract Background: Extracellular vesicles (EVs) have been isolated from various sources, including primary (show more...)Background: Extracellular vesicles (EVs) have been isolated from various sources, including primary and cultured cell lines and body fluids. Previous studies, including those conducted in our laboratory, have reported the stability of EVs under various storage conditions. Methods: EVs from human whole saliva were separated via size-exclusion chromatography. To simulate the effects of gastric or intestinal fluids on the stability of EVs, pepsin or pancreatin was added to the samples. Additionally, to determine the effect of bile acids, sodium cholate was added. The samples were then subjected to western blotting, dynamic light scattering, and transmission electron microscopy analyses. In addition, the activity of dipeptidyl peptidase (DPP) IV retained in the samples was examined to monitor the stability of EVs. Results: Under acidic conditions, with pepsin mimicking the milieu of the stomach, the EVs remained stable. However, they partially lost their membrane integrity in the presence of pancreatin and sodium cholate, indicating that they may be destabilized after passing through the duodenum. Although several associated proteins, such as mucin 5B and CD9 were degraded, DPP IV was stable, and its activity was retained under the simulated gastrointestinal conditions. Conclusion: Our data indicate that although EVs can pass through the stomach without undergoing significant damage, they may be disrupted in the intestine to release their contents. The consistent delivery of active components such as DPP IV from EVs into the intestine might play a role in the efficient modulation of homeostasis of the signal transduction pathways occurring in the gastrointestinal tract. (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 saliva Sample origin Control condition Focus vesicles extracellular vesicle 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 Size-exclusion chromatography (non-commercial) Filtration Ultrafiltration Protein markers EV: TSG101/ mucin 5B/ CD63/ CD81/ HSP90/ Alix/ Flotillin1/ IgA/ DPP IV/ HSP70/ CD9 non-EV: apolipoprotein B-100/ Albumin/ apolipoprotein A-1 Proteomics yes Show all info Study aim Function/Identification of content (omics approaches)/Technical analysis comparing/optimizing EV-related methods Sample Species Homo sapiens Sample Type saliva Separation Method Filtration steps > 0.45 µm, 0.22µm or 0.2µm Ultra filtration Cut-off size (kDa) 100 Membrane type Cellulose acetate Size-exclusion chromatography Total column volume (mL) 88 Sample volume/column (mL) 2 Resin type Sephacryl S-500 Characterization: Protein analysis Protein Concentration Method BCA Western Blot Antibody details provided? Yes Antibody dilution provided? Yes Lysis buffer provided? Yes Detected EV-associated proteins CD9/ CD63/ DPP IV/ mucin 5B/ IgA/ HSP90/ TSG101/ HSP70/ Alix/ CD81 Not detected EV-associated proteins Flotillin1 Detected contaminants apolipoprotein A-1/ apolipoprotein B-100/ Albumin Proteomics database Yes: Characterization: RNA analysis RNA analysis Type RNA sequencing;Capillary electrophoresis (e.g. Bioanalyzer) Database Yes Proteinase treatment No RNAse treatment No Characterization: Lipid analysis No Characterization: Particle analysis DLS Report type Mean Reported size (nm) 40 EM EM-type Transmission-EM Image type Close-up, Wide-field Report size (nm) 40

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EV-TRACK ID	EV200078
species	Homo sapiens
sample type	saliva
condition	Control condition
separation protocol	Size-exclusion chromatography (non-commercial) Filtration Ultrafiltration
Exp. nr.	1
EV-METRIC %	75