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. 2021 Oct 15;12:748706. doi: 10.3389/fmicb.2021.748706

FIGURE 1.

FIGURE 1

Outer membrane vesicle (OMV) entry into bone marrow-derived macrophages (BMDM) by fluorescence microscopy. BMDM seeded on cover glasses were co-incubated with Francisella tularensis-derived OMV (Ft-OMV) for the indicated time, fixed, and immunostained for fluorescence microscopy (A) and stimulated emission depletion (STED) microscopy (B). (A) Ft-OMV were visualized by immunostaining with purified immune rabbit polyclonal anti-F. tularensis serum, followed by Alexa FluorTM 488 anti-rabbit immunoglobulin G (IgG) (green signal). Actin was stained with phalloidin-TRITC (red signal) and cell nuclei with DAPI (blue signal). Arrows point to the internalized OMV. Untreated BMDM are shown as control. The accumulation of OMV signals around the nucleus is observed in the latest interval. The samples were observed using a ×60 objective. (B) Ft-OMV were stained with the anti-F. tularensis lipopolysaccharide (LPS) antibody followed by Alexa FluorTM 555 anti-mouse IgG (green) and the BMDM membrane with the anti-MHC class II antibody followed by Alexa FluorTM 488 anti-rat IgG (red). OMV alone are shown on the upper left. Shown are the maximal intensity projections of example cells from three independent experiments. Scale bar, 2 μm.