*3.1. EVs Secreted by Airway Epithelial Cells Are Mainly Released at the Apical Cell Surface*

To confirm that the isolated particles were indeed EVs, and to compare numbers and composition of apically versus basolaterally released EVs, NanoFCM analysis and bead-based flow cytometry with staining for characteristic tetraspanin surface markers of EVs were performed. Particle concentrations and size profiles showed significant differences in NanoFCM analysis, depending on their isolation side. Generally, epithelial cells secreted significantly larger quantities of particles at the apical cell surface compared to the basolateral side. NanoFCM analysis showed higher particle concentrations in apically obtained EV samples (Figure 2A). This finding was further confirmed by the results of protein concentration analyses, as depicted in Figure 2B. Furthermore, the size of particles retrieved from the apical cell side wash corresponded to the typical size range of exosomes with a mean median size of 75 nm (62–95 nm). Contrarily, particles isolated from the basolateral side were considerably larger in size with a mean median size of 169 nm (140–192 nm), consistent in size with microvesicles rather than with exosomes (Figure 2C,D). No differences in concentration or size range were observed between particles from healthy and asthmatic subjects.

**Figure 2.** EV characteristics in apically and basolaterally secreted vesicles. (**A**), Particle concentration after EV isolation analyzed by NanoFCM. (**B**), Protein concentration in EV isolates measured by NanoDrop Protein A280. (**C**), Median particle diameter and (**D**) size distribution of EVs isolated from the apical or basolateral side of bronchial epithelial cell cultures from healthy and asthmatic subjects analyzed by NanoFCM. Bars represent mean ± SD; dots indicate individual samples. \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001. EV—extracellular vesicle; n.s.—not significant.
