Linical Chemistry, and Vesicle Observation Center, Academic Health-related Center, University of Amsterdam, Amsterdam, The Netherlands, Amsterdam, The Netherlands; 4Department of Biomedical Engineering and Physics, and Vesicle Observation Center, Academic Health-related Centre on the University of Amsterdam, Amsterdam, The NetherlandsBackground: Transmission electron microscopy (TEM) is often a high-resolution imaging technique capable to distinguish extracellular vesicles (EVs) from similar-sized non-EV particles. Nevertheless, TEM sample preparation protocols are diverse and have in no way been compared directly to every other. In this study, we evaluate normally applied damaging staining protocols for their efficacy to detect EVs.Background: One of several major barriers in EV study will be the existing limitations of analytical tools for the characterization of EVs on account of their modest size and heterogeneity. EVs span a variety as modest as 50 nm to couple of microns in diameter. Not too long ago, flow cytometers have already been adapted to combine light scatter measurements from Dengue Virus Non-Structural Protein 5 (NS5) Proteins web nanoparticles with fluorescent detection of exosome markers. Nonetheless, the small-size of exosomes tends to make particular detection above background levels tough mainly because significant populations of small diameter vesicles (5000 nm) are also small for classic visualization technologies. Also, fluorescent surface marker detection is limited due to the lowered number of epitopes readily available to detect on a single particle. Approaches: To improved characterize these modest vesicles, we have created a label-free visible-light microarray imaging strategy termed Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows enumeration and sizing of person nanovesicles captured on the sensor which has been functionalized with an array of membrane protein precise capture probes. Additionally, we combined fluorescence detection with light scatter readout to co-localize multiple markers on individual EVs captured around the sensor surface. The fluorescence sensitivity was measured employing fluorescent polystyrene nanoparticles with diameters of 2000 nm, corresponding to 18010,000 fluorescein equivalent units. The calculated fluorescence detection limit approaches single fluorescence sensitivity. SP-IRIS technology requires a sample volume of 500 using a detection limit of five 105 particles/mL. Outcomes: To demonstrate the utility in the SP-IRIS detection technique we studied EV heterogeneity from 3 unique pancreatic cancer cell lines (Panc1, Panc ten.05 and BxPC3) by arraying the surface with antibodies against CD81, CD63, CD9, Epcam, EGFR, Tissue Element, Epcam, MHC-1, MHC-2 and Mucin-1. Additionally, to demonstrate the applicability on the SP-IRIS technologies for liquid biopsy we demonstrated detection of pancreatic cancer derived exosome spiked-in into human plasma. Summary/Conclusion: The SP-IRIS direct-from-sample high-throughput strategy could improve standardization of exosome preparations and facilitate translation of exosome-based liquid biopsies.Saturday, 05 MayLBS07: Late Breaking Poster Session Repair and Signalling Chairs: Costanza Emanueli; Geoffrey DeCouto Place: Exhibit Hall 17:158:LBS07.Exercise-induced muscle harm, extracellular vesicles and microRNA Jason Lovett; Peter Durcan; Kathy Myburgh Stellenbosch University, Stellenbosch, South AfricaBackground: Extracellular vesicles (EVs) are nano-sized (30000 nm) mediators of TAO Kinase 3 Proteins Species intercellular communication. EVs are steady and abundantly present in biofluids for instance.
