Figure 3

Visualization of intracellular S. aureus within (A) macrophages and (B-D) osteoblasts. Osteoblasts and macrophages were infected with S. aureus at an MOI of 500:1 for 2 h. (A and B) S. aureus was stained with FITC before infection. Infected osteoblasts and macrophages were fixed, blocked, stained first with primary antibody to S. aureus surface protein A, and then secondary antibody conjugated to Cy-5. The nuclei of the macrophages were additionally stained with DAPI. Visualized at (I) 488 nm, (II) 633 nm, and (III) 405 nm. (IV) Merged images of (I), (II), and (III). As a result, intracellular S. aureus is shown in green (FITC) and extracellular S. aureus is co-localized with both red (Cy-5) and green (FITC). (C) Z-stack sections were used to confirm that all live S. aureus was inside osteoblasts as determined by the X-Y planes. Live S. aureus are green (Syto 9) and dead S. aureus are red (PI). Osteoblasts were infected with Syto 9-labeled S. aureus, then extracellular bacteria were killed with gentamicin and washed. Osteoblasts were detached from the wells and stained with PI. Approximately 20 cells (randomly selected) were examined. (D) Confirmation of intracellular S. aureus within osteoblasts using TEM.