Labeling and Flow Cytometry of Extracellular Vesicles

The isolated EVs were labeled with CellTrace™Violet (Cat. No. C34557, Thermo Fisher Scientific). The labeling procedure was performed as suggested by the manufacturer. Briefly, the isolated EVs were incubated with the CellTrace reagent at a final concentration of 5 µM for 20 min at 37°C. The mixture was diluted with PBS or the FACS buffer and subjected to flow cytometry analysis.
Flow cytometry was performed using a CytoFLEX S Flow Cytometer with the laser configuration of V2B2Y3R2 and operated by the CytExpert Software v2.2 (Beckman Coulter, Brea, CA). The violet side scatter (VSSC) detector configuration was set for the detection of EVs [30 (link)].