Membrane Fusion Assay for S. pneumoniae EVs

To assess membrane fusion using S. pneumoniae EVs and J774A.1 macrophage cells, we essentially followed procedures described earlier [19 (link)]. Briefly, EVs were labeled with 1 mg/mL rhodamine isothiocyanate B-R18 (Molecular Probes) for 1 h at room temperature. An unlabeled probe was removed by centrifugation at 100,000× g (60 min, 4 °C). After washing with PBS, labeled EVs were resuspended in 1 mL PBS. Subsequently, the host cell plasma membrane was labeled for 1 h prior to the incubation with EVs with 8 mg/mL FITC-conjugated cholera toxin B subunit (CtxB) (Sigma-Aldrich). Then, labeled EVs were added in a 1:4 dilution in the wells and incubated for 30 min at 37 °C. When applicable, 10 mg/mL Filipin III was added 30 min prior to the addition of EVs. After incubation with EVs, cell samples were analyzed by confocal microscopy as described above.