Academic Journal
Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies
| العنوان: | Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies |
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| المؤلفون: | Benedikter, Birke J., Bouwman, Freek G., Vajen, Tanja, Heinzmann, Alexandra C. A., Grauls, Gert, Mariman, Edwin C., Wouters, Emiel F. M., Savelkoul, Paul H., Lopez-Iglesias, Carmen, Koenen, Rory R., Rohde, Gernot G. U., Stassen, Frank R. M. |
| المصدر: | Benedikter , B J , Bouwman , F G , Vajen , T , Heinzmann , A C A , Grauls , G , Mariman , E C , Wouters , E F M , Savelkoul , P H , Lopez-Iglesias , C , Koenen , R R , Rohde , G G U & Stassen , F R M 2017 , ' Ultrafiltration combined with size exclusion chromatography efficiently isolates extracellular vesicles from cell culture media for compositional and functional studies ' , Scientific Reports , vol. 7 , 15297 . https://doi.org/10.1038/s41598-017-15717-7 |
| سنة النشر: | 2017 |
| المجموعة: | Maastricht University Research Publications |
| مصطلحات موضوعية: | HUMAN PLASMA, EXOSOMES |
| الوصف: | Appropriate isolation methods are essential for unravelling the relative contribution of extracellular vesicles (EVs) and the EV-free secretome to homeostasis and disease. We hypothesized that ultrafiltration followed by size exclusion chromatography (UF-SEC) provides well-matched concentrates of EVs and free secreted molecules for proteomic and functional studies. Conditioned media of BEAS-2B bronchial epithelial cells were concentrated on 10 kDa centrifuge filters, followed by separation of EVs and free protein using sepharose CL-4B SEC. Alternatively, EVs were isolated by ultracentrifugation. EV recovery was estimated by bead-coupled flow cytometry and tuneable resistive pulse sensing. The proteomic composition of EV isolates and SEC protein fractions was characterized by nano LC-MS/MS. UF-SEC EVs tended to have a higher yield and EV-to-protein rate of purity than ultracentrifugation EVs. UF-SEC EVs and ultracentrifugation EVs showed similar fold-enrichments for biological pathways that were distinct from those of UF-SEC protein. Treatment of BEAS-2B cells with UF-SEC protein, but not with either type of EV isolate increased the IL-8 concentration in the media whereas EVs, but not protein induced monocyte adhesion to endothelial cells. Thus, UF-SEC is a useful alternative for ultracentrifugation and allows comparing the proteomic composition and functional effects of EVs and free secreted molecules. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| DOI: | 10.1038/s41598-017-15717-7 |
| الاتاحة: | https://cris.maastrichtuniversity.nl/en/publications/76796600-e514-4c91-8356-1a408425c2c8 https://doi.org/10.1038/s41598-017-15717-7 |
| Rights: | info:eu-repo/semantics/openAccess |
| رقم الانضمام: | edsbas.74521D4C |
| قاعدة البيانات: | BASE |
| DOI: | 10.1038/s41598-017-15717-7 |
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