Fig. 1

Successful isolation of S. aureus M5512B extracellular vesicles (SaEVs) and in vitro generation of bovine monocytes derived macrophages (boMdM). (a) TEM images showing heterogenic populations of SaEVs. Black arrows point to representative SaEV structures. (b) TEM images confirm the absence of SaEVs in the EV-depleted supernatant (non-EV secretions). (c) Particle concentration and size distribution of SaEVs used for the in vitro stimulation. (d) Full WB membrane shows alpha-toxin (Hla) in SaEVs (3–5) but not in non-EV secretions (6,7), TSB medium without S. aureus (1), or non-processed S. aureus medium (2). In samples 2–5, a strong band around 50 kDa is observable, which might indicate the presence of an immunoglobulin-binding protein that binds to the Fc regions of both the primary and secondary antibodies²². L: protein size ladder. (e) Diff-Quik staining of the in vitro derived boMdM used for the experiments showed the typical flattened, adherent shape with an amoeboid appearance macrophage morphology. (f) Confocal imaging confirmed that boMdM were positively stained with CD14, commonly considered a macrophage marker. (g) Negative control staining included only the secondary antibody.