deoB is important for entry of F. tularensis LVS. (A) Primary human macrophages (MΦs) or dendritic cells (DCs) were infected with either LVS, 1664d, or 1664d/pFTL_1664 in microtitre plates. After a two-hour incubation, wells were treated with gentamicin to kill extracellular bacteria, followed by vigorous washing. Subsequently, phagocytic cells were lysed and serial dilutions of lysates were plated for CFU enumeration. Data are mean ± SEM of triplicate wells within one experiment and are representative of four (primary human macrophages) or two experiments (dendritic cells) performed using cells from separate donors. Following log transformation, differences in CFU between LVS and 1664d were determined by a Student's t-Test in which P = 0.00005 and 0.003 for MΦs and DCs, respectively. (B) Bacteria were stained with the fluorescent green stain, Syto-9 prior to infection. After a two-hour incubation with RAW 264.7 cells, extracellular bacteria were washed away and cells were analyzed under brightfield (BF) and fluorescence (Fluor.) microscopy. The exposure time was extended to enhance sensitivity of detecting the low numbers of 1664d within the RAW 264.7 cells, leading to some background fluorescence. Fluorescence images were initially captured in grayscale and pseudocolored using Adobe Photoshop. Data displayed are representative of duplicate experiments. Scale bar = 50 μm. (C) Human embryonic kidney 293 (HEK-293) cells were infected similarly to the phagocytic cells in panel A. Data are mean ± SEM of triplicate wells within one experiment and are representative of three experiments. Following log transformation, differences in CFU between LVS and 1664d were determined by a Student's t-Test in which P = 0.0004.