Browsing by Subject "density gradient centrifugation"

Tiivistelmä - Referat - Abstract Background. Platelets are known to contain ample amounts of brain derived neurotrophic factor. Previous spectrophotometric studies carried out in Pia Siljander’s lab have shown that BDNF is secreted from activated platelets packed in extracellular vesicles. For this project we wanted to 1) confirm that BDNF really is secreted in extracellular vesicles (EVs)2) find out how the choice of agonist affected the BDNF cargo of the platelet derived EVs, and 3) find out if the BDNF is packed into EVs of certain densities rather than others. Methods. The platelets were isolated from platelet concentrates by size exclusion chromatography. The isolated platelets were then activated by thrombin and collagen co-stimulation (TC) and by Ca2+ionophore, respectively. The platelet activation produced extracellular vesicles (PEVs) which were isolated by differential ultracentrifugation. The isolated PEVs were then analysed by flow cytometry, ELISA and Western blot for EV typical membrane surface proteins and for their BDNF content. As we were interested finding out whether BDNF is enriched in PEVs to certain populations, density gradient centrifugation was performed. These samples were also analysed by Western blot and by ELISA. The size distribution and concentration of PEVs in all samples was analysed by Nanoparticle tracking analysis. Results and conclusions. This study confirmed that platelets secrete PEVs as a response to agonists. PEVs with higher BDNF concentration were produced using TC co-stimulation as compared to PEVs derived from the Ca2+ionophore. The result implies that BDNF is actively packed into PEVs for instance as a thrombogenic response. Based on the density gradient results it seems that BDNF was packed into certain population of PEVs with a density between 1.112 g ml-1 and 1.132 g ml-1 corresponding to a particle diameter of less than 500 nm. The finding that BDNF is actively packed into TC co-stimulation derived PEVs of a certain population is interesting from a theragnostic point of view, since EVs are likely to be key players in the development of new cell-based therapies. Had there been more time, it would have been interesting to optimize both the density gradient protocol and the ELISA analysis. This optimization of methods would make the process more efficient, less prone to sample loss, not to mention that there would be less intra-assay variation.