FIG 5.

Sera from animals immunized with AuNP vaccines promoted bacterial opsonophagocytosis and killing by mouse primary macrophages. (A) Mouse primary macrophages were infected with bacteria in the presence or absence of immune sera (10% of final volume) from immunized animals with each vaccine formulation (n = 5) at an MOI of 10 for 2 h at 37°C. Sera from naive mice (adjuvant only) served as controls. To visualize bacterial uptake by macrophages, 2 h after infection cells were stained as described for Fig. 4. (B) To assess bacterial killing, infected macrophages with bacteria treated with immune sera were permeabilized with 0.1% saponin and stained using LIVE/DEAD BacLight. SYPRO-9 (green) will enter all bacterial cells, but only membrane-compromised bacteria will integrate PI (red) into DNA, giving a higher red intensity. Panels below each group represent magnifications (10×) of the images on top. Images were taken using an Olympus BX51 upright fluorescence microscope (60×) and processed using Image J software (37). Scale bars, 25 μm.