Optimizing Extracellular Vesicle Biotinylation Analysis

The biotinylation efficiency of EVs and the optimization of electroporation were analyzed by using an Apogee A‐50 Micro Flow Cytometer (Apogee Flow Systems) equipped with 375, 405, 488, and 638 nm lasers. The reference beads (ApogeeMix, Apogee Flow Systems) composed of a mixture of nonfluorescent silica beads (Si, refractive index ≈1.42) with diameters of 180, 240, 300, 590, 880, 1300, and 110 nm, 500 nm green fluorescent latex spheres (polystyrene, refractive index ≈1.59) were used to set the thresholds for light scattering and help to gate sEVs and lEVs. The tubing was washed with double‐distilled water after each sampling. Analysis was performed at a flow rate of 1.5 µL min⁻¹ using a 150 µL sample volume for at least total 3 × 10⁵ particles.