R from technical issues, and classical nanoparticle tracking analysis (NTA) permits quantification and size determination of particles, but fails to discriminate in between EVs, lipids and protein aggregates. Fluorescence-based NTA (FL-NTA) is an emerging technique for counting and phenotyping of EVs. EVs is usually fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. We are presently establishing a differential FL-NTA approach employing particular antibodies against surface markers in analogy to cell flow cytometric evaluation. Methods: EVs from umbilical cord mesenchymal stromal cells (UCMSCs) were isolated by a tangential flow filtration/ultracentrifugation protocol with or devoid of subsequent size exclusion chromatography. EV preparations had been stained with AlexaFluor 488-conjugated certain antibodies or corresponding isotype controls. Quantity and size of particles in regular scattering light mode (N mode) versus fluorescence mode (FL mode, laser wavelength 488 nm) was measured making use of ZetaView Nanoparticle Tracking Analyzer (Particle Metrix). Final results: All UC-MSC-EV preparations have been discovered optimistic for typical EV marker proteins and negative for MHC I. Further 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 of your percentage of EV marker protein-positive nanoparticles inside a mixed particulate solution. We aim to expand our set of markers to other MSC-EV optimistic and negative surface marker proteins to be able to establish FL-NTA-based surface marker profiling as an more technique for quantifying EVs. Funding: This perform was supported by project EXOTHERA (funded by the European Regional Development Fund and Interreg V-A ItaliaAustria 2014-2020).PS09.Imaging flow cytometry: a potent system to recognize 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, Department 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 with the extracellular vesicle content in mixed particulate solutions Karin Pachler1; Alexandre Desgeorges1; Christina Complement Receptor 1 Proteins manufacturer Folie1; Magdalena Mayr1; Heide-Marie Binder1; Eva Rohde2; Mario Gimona1 GMP Unit, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Healthcare University Salzburg, Salzburg, Austria; 2 GMP Unit, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS) and University Institute for Transfusion Medicine, Paracelsus Carbonic Anhydrase 14 (CA-XIV) Proteins medchemexpress Health-related University Salzburg, Salzburg, AustriaBackground: Although distinct extracellular vesicle kinds have already been defined with regards to their cellular origin, for now, exosomes can hardly been discriminated from modest microvesicles or other compact EV types. You will find hardly any methods offered, now, enabling to discriminate unique EV-types of comparable sizes. Not too long ago, we’ve got optimized imaging flow cytometry for the single EV detection and characterization of tiny EVs (7050 nm) [1]. Upon extending our imaging flow cytometric ana.
