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Browsing by Subject "nanoparticle tracking analysis"

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  • Lindelöf, Anna-Emilia (2019)
    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.
  • Puutio, Johanna (2020)
    Extracellular vesicles (EVs) are phospholipid bilayer-enclosed nanoparticles that are secreted by eukaryotic and prokaryotic cells. EVs carry macromolecules and signalling molecules to adjacent cells and play an important role in intercellular communication under both pathologic and homeostatic conditions. Therefore, they have become of significant interest for their therapeutic, diagnostic and prognostic potential. EVs are small and highly heterogeneous in size, shape, cargo and membrane composition, posing several challenges for establishing analytical and clinical guidelines. Therefore, EV research requires standardized and robust methods for their separation and characterization. In this study physical and immunochemical methods were employed to characterize human platelet-derived EVs (pEVs) generated from platelets activated with different external biochemical stimuli. The platelet-activating effect of the pro-inflammatory S100A8/A9 protein complex and a combination of thrombin and collagen were studied with nano flow cytometry. The size distribution of pEVs was studied with nanoparticle tracking analysis (NTA) and asymmetrical flow field-flow fractionation (AF4), which represents a newly emerging method on the EV field. Finally, fluorescent labelling and co-localization analysis were employed to characterize membrane marker composition of pEVs and assess its usefulness as an analytic tool for EV research. We succeeded in providing new hints towards meaningful discoveries in platelet biology by characterizing the way platelets respond to inflammatory and hemostatic signals by shedding pEVs. When platelet activation markers are characterized with flow cytometry, the S100A8/A9 protein appeared to cause a shift in membrane activation markers when compared to the thrombin- collagen mix and the baseline control. Increased TLT-1 translocation and decreased integrin αIIbβ3 expression on pEV surfaces suggests that S100A8/A9 induced pEV secretion through differently packed platelet α-granules, rather than from the plasma membrane. An increase in TLT-1 expression compared to decreased P-selectin and αIIbβ3 suggests that S100A8/A9 stimulation shifts platelet phenotype towards secretion rather than aggregation. A protocol for small pEV separation with AF4-MALS was set up. With this method, subtle differences between small pEV populations were seen that were not distinguishable with NTA or flow cytometry. When investigated with AF4-MALS, S100A8/A9 induced pEVs appeared larger than those produced with thrombin- collagen activation. The mean particle sizes of the pEV populations obtained from activated platelets were generally also larger than those produced without an activator. We tested novel methods to detect subtle differences in small EV population sizes that are easily missed with conventional methods due to their technical limitations. A well-optimised AF4 protocol can detect different pEV subpopulations and is a promising tool for EV. In the future, when AF4 is combined with a MALS detector and a fraction collector, nanoimaging of fluorescently labelled EVs could be combined with it as a downstream application to obtain information on their versatile biological functions.
  • Stendahl, Annie (2022)
    Abstract Faculty: Faculty of Biological and Environmental Sciences Degree programme: Genetics and Molecular Biosciences Study track: Molecular and Analytical Health Biosciences Author: Annie Stendahl Title: Measurement repeatability of flow cytometry and nanoparticle tracking analysis for optimization of extracellular vesicle measurements Level: Master’s thesis Month and year: 11/2022 Number of pages: 92 Keywords: extracellular vesicles, repeatability measurements, metrology, traceability, flow cytometry, nanoparticle tracking analysis, reference material, METVES Supervisor or supervisors: Virpi Korpelainen, Katariina Maaninka and Pia Siljander Collaborative partner: VTT Technical Research Centre of Finland Ltd. Where deposited: E-thesis Extracellular vesicles (EVs) are lipid bilayer-enclosed vesicles secreted by all cells, containing variable cargo from nucleic acids and proteins to carbohydrates, metabolites, and lipids. EVs are considered to be involved in many physiological and pathological cell functions. Due to their presence in biofluids hence enabling semi-invasive liquid biopsies, EVs have indicated great promise for utilization as biomarkers in clinical settings. The innate properties of EVs and their cargo could also be harnessed into therapeutic use. However, the current methods and reference materials for determining EV concentration and size have not yet achieved the metrological level of repeatability and traceability, which is needed for EV measurements to be utilized in clinical settings. The aim of this thesis project was to evaluate repeatability of the methods typically used for EV quantification and size determination, flow cytometry (FCM) and nanoparticle tracking analysis (NTA). The repeatability was analyzed with reference material made of hollow organosilica beads and biological EV test samples, both developed in an ongoing EU metrology-project METVES II for EVs. A similar biological EV test sample was also prepared as part of the thesis project. Finally, the repeatability measurements were conducted with calibration beads recommended by the instrument manufacturers. The calibration beads gave repeatable results with FCM and one of the two NTA instruments tested, but neither the reference beads nor the biological EV test samples produced repeatable results to enable determination of repeatability. However, valuable understanding was gained on what can be optimized during the measurements and operation of the instruments to generate more repeatable results with FCM and NTA in EV analysis. Prior knowledge of both the sample type and method used for measuring would enable optimization of the measurement and instrument operation. Whether the aim is EV quantification or size determination, instrument errors and bias could then be minimized by adjusting the settings according to sample type. Furthermore, EV quantification and size determination would benefit from combining different methods to ensure more reliable and repeatable results. It is clear that more research needs to be done, for i.e., the tested reference beads need to be further developed to be established as EV reference material and enabling standardization of EV measurements. Standardizing EV quantification and size determination is required to achieve metrological repeatability and ultimately, traceability, and thus for EVs to be utilized in clinical settings as biomarkers or therapeutic use.