A Multiplex Flow Cytometric Approach to Define Molecularly Distinct Extracellular Vesicle Subsets

ABSTRACT: Extracellular vesicles (EVs) are molecularly very heterogeneous, and their characterization at the single-particle level is technically challenging. Existing approaches, such as nanoparticle tracking analysis, fluorescence microscopy, and nano-flow cytometry, provide important insights but often lack the flexibility to detect multiple molecular markers simultaneously. Here, we describe an optimized workflow for multiparametric EV phenotyping using a spectral flow cytometry instrument with enhanced small particle detection capacity. EVs were isolated from murine melanoma and melanocyte cell lines via size-exclusion chromatography and labeled with a fluorogenic membrane probe that enables robust, single EV detection. In this study, we systematically optimized staining conditions, EV concentrations, and fluorophore combinations for a 5-color antibody panel on single EVs. We show that single-particle flow cytometry can reliably detect and resolve multiple EV surface markers simultaneously. Data analysis by unsupervised clustering further enabled unbiased identification of distinct EV subsets, providing a practical approach for EV phenotyping in both research and clinical contexts.