*3.2. Differential Surface Marker Expression Profiles between Apically and Basolaterally Released EVs*

Bead-based flow cytometry using cocktails of antibodies directed towards the EVidentifying tetraspanin surface markers CD9, CD63, and CD81 for bead binding and detection did not reveal any significant differences between apical and basolateral EVs. However, when examining the expression of singular surface proteins, vesicles secreted to the basolateral side showed a higher expression of CD9 and CD81 compared to apically secreted vesicles, while for CD63, such a difference could not be observed (Figure 3A,B). When further looking at the subgroups, namely EVs from asthmatic and healthy subjects, these differences in marker expression depending on secretion side were similarly represented. However, CD9 expression by apical compared to basolateral EVs seemed to differ to a greater extent in the EVs of healthy subjects. When comparing the surface marker expression of EVs from healthy and asthmatic subjects, subtle distinctions in the expression of CD9 were observed on the basolateral side, but not for the other tetraspanins. No differences could be found in these groups for vesicles from the apical cell side (Figure 3C). This differential representation of selective marker proteins on EVs was generally suggestive for compositional differences, depending on the cell side of secretion.

**Figure 3.** Bead-based flow cytometry analysis of tetraspanins surface marker expression on EVs released by airway epithelial cells. Vesicles were captured using beads coated with a mixture of CD9-, CD63-, and CD81-specific antibodies. Specific secondary antibodies coupled with either CD9, CD63, or CD81, or a combination of all three, were used for detection. (**A**) The results of one representative experiment demonstrating tetraspanin expression on EVs isolated from apical and basolateral cell culture medium are shown. (**B**) Tetraspanin expression in EVs isolated from basolateral versus apical cell culture medium regardless of disease expression. (**C**) Comparison of the surface marker expression of apical and basolateral EVs in healthy and asthmatic subjects. n = 3 in each group, \* *p* < 0.05, \*\* *p* < 0.01. EV—extracellular vesicle; ns—not significant; RFU—relative fluorescent units.
