Iological effect in humans. Funded by the Dutch Arthritis Association RF 12-2-201.Heinrich Resch8, Susanne Wolbank4, Heinz Redl4, Pidder Jansen-Durr9, ?Peter Pietschmann10, Regina Grillari-Voglauer2 and Johannes Grillari1 Department fur Biotechnologie, University of All-natural Resources and Life ?Sciences, Vienna; 2Evercyte GmbH, Vienna, Austria; 3Department fur ?Biotechnologie, Evercyte GmbH, Vienna, Austria; 4Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria; 5Institute of Biomedical Aging Investigation, Austrian Academy of Sciences, Innsbruck, Austria; 6 Children’s Cancer Analysis Institute (CCRI), St. Anna Kinderkrebsforschung, Vienna, Austria; 7Department of Gerontology and Geriatrics, Leiden University Health-related Center, Leiden, The Netherlands; 8Medical University, Vienna, Austria; 9 Austrian Academy of Sciences, Innsbruck, Austria; 10Department of Pathophysiology, jir.2014.0227 Health-related University, Vienna, Austria; 11Department of Biotechnology, University of Organic Resources and Life Sciences Vienna, Vienna, AustriaO8C-Human breast milk contains several subpopulations of extracellular vesicles carrying immune modulatory proteins Marijke I. Zonneveld1,two, Martijn J.C. van Herwijnen1, Alain Brisson3, Maarten Altelaar4, Frank A. Redegeld2, Johan Garssen5, Marca H.M. Wauben1 and Esther N.M. Nolte – ‘t HoenBiochemistry Cell Biology, Universiteit Utrecht, Utrecht, The Netherlands; Pharmaceutical Sciences, Universiteit Utrecht, Utrecht, The Netherlands; three Molecular Imaging and Nano Bio Technology, University of Bordeaux, Talence, France; 4Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Investigation and Utrecht Institute for Pharmaceutical Sciences, Utrecht, The Netherlands; 5Nutricia Analysis Centre for Specialized Nutrition, Utrecht, The Netherlands2Introduction: Extracellular vesicles (EV) with immune modulatory properties in human breast milk may perhaps be involved in instruction in the neonatal immune program. Previously, we created a GDC-0810 chemical information density gradient-based protocol for efficient and trusted recovery of EV from breast milk. Right here, we established a milk storage protocol permitting isolation of naturally occurring EV, although avoiding the generation of contaminating EV induced by storage. Furthermore, we identified and characterized a variety of subpopulations of breast milk EV. Techniques: EV were isolated from fresh human milk or stored cell-free milk supernatant. EV subsets and non-floating complexes were characterized by high-resolution flow cytometry, cryo EM, proteomics and western blotting. Benefits: Cold storage at 548C of unprocessed milk led to milk cell death. By spiking human milk with murine cells, we discovered that cell death during cold storage led to contamination in the milk EV population with vesicles generated in the course of storage. Storage of fresh milk samples right after immediate removal of cells and fat prevented this contamination, whereas the EV recovery was comparable to fresh milk. Evaluation by high-resolution flow cytometry revealed that quantities of EV and their distribution over the density gradient have been comparable in milk of diverse donors. The predominant milk EV subpopulations resided within the 1.12?.18 and 1.18?.21 g/ml density fractions. Analysis by high-resolution flow cytometry and cryo EM showed that each EV populations had been heterogeneous in forward journal.pone.0169185 light scattering, size, and annexin V labelling. Furthermore, lengthy tubular structures have been identified within the 1.12?.18 g/ml fraction. By c.