Onomy of Hungary [VEKOP-2.three.2-16-2016-00002 and VEKOP-2.three.350160016].Background: Nanoparticle tracking analysis (NTA) of bionanoparticles, for instance EVs, vesicles or liposomes, is definitely an effective approach for quantification of size and total concentration. With fluorescence detection choice, F-NTA enables the precise quantification of subpopulations of biomarkers on single particle level. Traditionally, samples are analysed applying only one particular laser wavelength. For the first time, we show phenotyping of EVs by a NTA instrument equipped with two laser sources, 405 nm and 488 nm, enabling rapid evaluation of biomarker concentration or ratios. Techniques: EVs had been derived from cell line and plasma respectively and isolated and purified by ultracentrifugation, Zika Virus Non-Structural Protein 5 Proteins MedChemExpress tangential flow filtration or size exclusion MMP-12 Proteins site chromatography. For the determination of vesicle content material, protocols for many plasma membrane dyes had been developed and optimized for NTA detection. Various antibodies have been evaluated for EV characterization and protocols were optimized for NTA detection. Benefits: Switching between scatter and fluorescence mode allows quantification of vesicle content. The efficiency depending on protocol and dye for example PKH67, DiO and CMG are compared. Impact of bleaching was minimized due to fast acquisition. A number of fluorescently labeled antibodies for detection of CD63, CD81 and CD9 have already been evaluated. Total concentration also as biomarker ratios are presented as function of origin and purification of EVs. Summary/Conclusion: Phenotyping of EVs derived from cell line and plasma was performed by multiwavelength NTA applying 405 nm and 488 nm for excitation. Alignment-free switching involving excitation wavelengths allows quantification of biomarker ratios around the similar sample within minutes decreasing measurement time and precious sample quantity.LBT01.Comparative analyses of exosome isolation procedures from distinct biofluids T ia Soares Martins1; JosCatita2; Ilka Martins Rosa1; Odete A. B. da Cruz e Silva1; Ana Gabriela Henriques1 iBiMED – Institute of Biomedicine, Aveiro, Portugal; Gondomar, Gondomar, PortugalParalab SA,LBT01.Low-density lipoprotein associates with extracellular vesicles by means of apolipoprotein B Barbara W Sodar1; Krisztina P zi1; Tam Visnovitz1; Krisztina V Vukman1; a P linger1; p Kov s1; Eszter T h1; Hargita Hegyesi1; nes Kittel2; S a T h1; Edit BuzasDepartment of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary; 2Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, HungaryBackground: We’ve got shown not too long ago that low-density lipoprotein (LDL) co-isolates with extracellular vesicles (EVs) derived from blood plasma as well as the supernatant of platelet concentrates. Moreover, we identified that with current isolation protocols, EVs and LDL can not be separated. By transmission electron microscopy we also demonstrated the association of EVs with LDL in vitro.Background: Exosomes are present in different physique fluids and may cross blood-brain barrier, which enhances their prospective as drug-delivery targets but in addition as diagnostic tools. Indeed, these nanovesicles is often a resource for proteomic, lipidomic and genetic biomarkers. Nevertheless, exosome isolation from different biofluids is usually a challenge. Differential ultracentrifugation could be the most commonly made use of system although it’s laborious and not sufficient for large-scale clinical research; hence alternative solutions are urgently needed. Other methodologies happen to be addresse.
