On-column staining of microbead-isolated extracellular vesicles (EVs) enables effective detection and immunophenotyping of EV populations on conventional flow cytometers 4381

Abstract Description Interest in extracellular vesicles (EV) has grown tremendously as they have been found to play a critical role in cell communication and regulation, but their isolation and analysis remains a challenge. Flow Cytometry is a powerful tool for analyzing complex cell populations however, the detection and analysis of EVs fall near or below the detection limits of conventional flow cytometers. Here we demonstrate that microbead isolation of EVs simplifies their labeling and washing processes as well as enhancing their detectability using conventional flow cytometers. EVs from T Cell TransAct™-stimulated and non-stimulated peripheral blood mononuclear cells (PBMCs) were magnetically isolated using Miltenyi Biotec Pan EV Isolation microbeads. While on the isolation column, bound EVs were labeled with antibodies and EV-identifying stains. After elution, the samples were directly analyzed on MACSQuant® Analyzer flow cytometers. Our results show that EVs from both unstimulated and stimulated cells can be detected above background levels. Changes in multiple EV populations were measured, and are consistent with large bead-based flow assays and previously published studies. Control detergent-lysis and serial dilutions confirmed that detected events represent unique membrane-bound entities. These findings suggest that on-column staining of microbead-isolated EVs effectively allows for the immunophenotyping of EV populations using conventional flow cytometry equipment. Topic Categories Technological Innovations in Immunology (TECH)