Figure 10.

S. aureus bMVs and their associated RNA cargo is delivered into wild-type macrophages. (A) Wild-type RAW 264.7 macrophages were treated with DiD-labeled bMVs (magenta) for 1 h. Macrophages were then fixed and permeabilized using saponin. S. aureus 5-EU RNA in macrophages was reacted and stained with the Click-iT Alexa Fluor 488 microscopy labeling kit (green), and counterstained with DAPI (nucleus; blue). Representative z-stack projections and their corresponding 3D reconstructions are shown for each of the 2 independent experiments that were performed. N = 64 stacks (top panel) and N = 64 stacks (bottom panel). (B) Wild-type RAW 264.7 macrophages were treated with DiD + SYTO RNA-labeled bMVs (green; bMV RNA & magenta; bMV lipids) for 1 h. Macrophages were then fixed, permeabilized, and stained with DAPI. Representative z-stack projections and their corresponding 3D reconstructions are shown for each of the 2 independent experiments that were performed. N = 40 stacks (top panel) and N = 64 stacks (bottom panel). Images demonstrate that bMV-associated RNA molecules can be delivered to macrophages and possibly released into the cytoplasm and peri-nuclear region after 1 h of co-incubation. All images were captured using a Zeiss LSM 880 equipped with Airyscan (63 × objective). Scale bars are indicated.