R from technical troubles, and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate involving EVs, lipids and protein aggregates. Fluorescence-based NTA (FL-NTA) is definitely an emerging system for counting and phenotyping of EVs. EVs is often fluorescently labelled with non-specific membrane markers or with antibodies especially recognizing EV surface Caspase Activator Formulation marker proteins. We’re at present establishing a differential FL-NTA approach employing specific antibodies against surface markers in analogy to cell flow cytometric analysis. Techniques: EVs from umbilical cord mesenchymal stromal cells (UCMSCs) had been isolated by a tangential flow filtration/ultracentrifugation protocol with or devoid of subsequent size exclusion chromatography. EV preparations have been stained with AlexaFluor 488-conjugated distinct antibodies or corresponding isotype controls. Amount and size of particles in regular scattering light mode (N mode) versus fluorescence mode (FL mode, laser wavelength 488 nm) was measured utilizing ZetaView Nanoparticle Tracking Analyzer (Particle Metrix). Benefits: All UC-MSC-EV preparations had been identified positive for common EV marker proteins and damaging for MHC I. More purification of EV preparations by size exclusion chromatography led to a greater percentage of EV marker protein-positive nanoparticles. Summary/Conclusion: Differential FL-NTA facilitates determination on the percentage of EV marker protein-positive nanoparticles inside a mixed particulate remedy. We aim to expand our set of markers to other MSC-EV good and adverse surface marker proteins to be able to establish FL-NTA-based surface marker profiling as an extra system for quantifying EVs. Funding: This function was supported by project EXOTHERA (funded by the European Regional Improvement Fund and Interreg V-A ItaliaAustria 2014-2020).PS09.Imaging flow cytometry: a potent approach to determine distinct subpopulations of small extracellular vesicles Michel Bremer1; Rita Ferrer-Tur1; AndrG gens2; Verena B ger3; Peter A. Horn3; Bernd Giebel3 Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany; 2Clinical Analysis Center, Division for Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, H sov en, Sweden; 3Institute for Transfusion Medicine, University Hospital Essen, Essen, GermanyPS09.Differential fluorescence nanoparticle tracking evaluation for enumeration of your extracellular vesicle content material in mixed particulate solutions Karin Pachler1; Alexandre Desgeorges1; Christina Folie1; Magdalena Mayr1; Heide-Marie Binder1; Eva Rohde2; Mario Gimona1 GMP Unit, ERβ Agonist custom synthesis Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Health-related University Salzburg, Salzburg, Austria; 2 GMP Unit, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS) and University Institute for Transfusion Medicine, Paracelsus Medical University Salzburg, Salzburg, AustriaBackground: Although various extracellular vesicle varieties have already been defined concerning their cellular origin, for now, exosomes can hardly been discriminated from small microvesicles or other little EV sorts. You can find hardly any methods accessible, now, enabling to discriminate various EV-types of comparable sizes. Recently, we’ve optimized imaging flow cytometry for the single EV detection and characterization of little EVs (7050 nm) [1]. Upon extending our imaging flow cytometric ana.