*2.5. Bead-Based Flow Cytometry*

EVs were detected by bead-based flow cytometry as previously described by Benedikter et al., with some adaptations [26]. Briefly, 4 μm aldehyde/sulphate latex beads at 3.5 × <sup>10</sup>8/mL (Thermo Fisher Scientific, Waltham, MA, USA) were incubated with 0.125 mg/mL of an equal mixture of three monoclonal antibodies (anti-human CD9 (clone M-L13), anti-human CD63 (clone H5C6), anti-human CD81 (clone JS-81); all antibodies were purchased from BD Biosciences, Franklin Lakes, NJ, USA) overnight in an MES buffer (Sigma Aldrich, St. Louis, MO, USA) on a shaker at 6500 rpm. The coated beads were stored in PBS containing 0.1% (*m*/*v*) glycine and 0.1% (*m*/*v*) sodium azide at 4 ◦C until use. Before use, the beads were washed with PBS containing 2% (*w*/*v*) bovine serum albumin (BSA). Then, 1 × 106 beads in 1 <sup>μ</sup>L were incubated overnight with 100 <sup>μ</sup>L of isolated EVs suspended in PBS at a concentration of 1.6 × 107 particles/mL based on NanoFCM measurements. Detection was performed as described previously, with either one or a mixture of the following phycoerythrin (PE)-labelled antibodies: anti-human CD9, anti-human CD63, and anti-human CD81 (same clones as above) [26]. Stained beads were suspended in 150 μL PBS and were analyzed using a BD FACSCanto II and FACS Diva v8.0.1 analysis software (BD Biosciences, Franklin Lakes, NJ, USA). To quantify the EV surface marker expression, relative fluorescence units (RFU) were calculated by multiplying the percentage of PE-positive beads with the median fluorescent intensity (MFI) of the positive bead population, as described by Benedikter et al. [26].
