Cardiocentro Ticino Foundation and Swiss Institute for Regenerative Medicine (SIRM) 1 article published in JoVE Biology Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media Carolina Balbi*1, Sara Bolis*1, Giuseppe Vassalli1,2,3, Lucio Barile1 1Cellular and Molecular Cardiology Laboratory, Cardiocentro Ticino Foundation, Switzerland, 2Molecular Cardiology Institute, Dept. of Cardiology, University of Zürich, 3Faculty of Biomedical Science, Università Svizzera Italiana, Switzerland The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.