Isolation and characterization of platelet-derived extracellular vesicles. A., Laitinen S., Gronholm M., Siljander P. Moreover, isolation of lipoprotein- and albumin-free EVs from blood plasma can be achieved by DGUC followed by BEC, however, on the expense of reduced EV yield.īind-elute chromatography density gradient ultracentrifugation exosomes extracellular vesicles iodixanol isolation plasma.Īatonen M. Conclusion: This is the first demonstration that DGUC is able to markedly reduce the lipoprotein content of EV isolates while it separates EVs with high efficiency. However, BEC decreased efficiency of EV isolation, and fibrinogen was still present in EV-rich fractions. Second-step purification by UC or SEC did not improve EV separation, while after BEC on HiScreen Capto Core 700 albumin and lipoprotein contamination were below detection limit in EV-rich fractions. However, 32.8 ± 1.5% of the total fibrinogen beta was found in this fraction. Only 1.4 ± 0.5% of LDL and chylomicron marker, 3.0 ± 1.3% of HDL marker, and 9.9 ± 0.4% of albumin remained in the EV-rich F6. Results: The highest band intensities of EV markers Alix, Tsg101 and CD81 were detected by Western blot in F6 of small-scale DGUC (61.5 ± 10.4% 48.1 ± 5.8% 41.9 ± 3.8%, respectively) at a density of 1.128-1.174 g/mL, where the presence of vesicles with a mean diameter of 38 ± 2 nm was confirmed by EM and DLS. Morphology and size distribution of particles were examined by dynamic light scattering and electron microscopy (EM). Efficiency and purity were assessed by Western blots. Fractions with the highest EV content were further purified by ultracentrifugation, size exclusion, or bind-elute chromatography. Ten fractions (F1-10) were collected from top to bottom. Density gradient ultracentrifugation was performed by loading plasma sample onto 50, 30, and 10% iodixanol layers and then centrifuged at 120,000 × g for 24 h. Methods: Samples of rat blood were centrifuged to remove cells, platelets, large EVs and protein aggregates without prior filtration. In accordance with this challenge, the aim of this study was to develop an isolation method in which to separate the majority of EVs from major impurities such as lipoprotein particles and the abundant plasma proteins albumin and fibrinogen. One challenging aspect to this research is the efficient isolation of high-purity EVs from blood plasma in quantities sufficient for in vivo experiments. Background: Extracellular vesicles (EVs) (isolated from blood plasma) are currently being extensively researched, both as biomarkers and for their therapeutic possibilities.
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