To determine whether the bacteria were affecting the viability of the endothelial cells, four methods were used. First, adherent and EDTA-lifted endothelial cells infected at an MOI of 10 were washed, then incubated with the vital dye CellTracker Green (CT-CMFDA, 10 mM) plus DAPI (0.02 mg/ml) (Molecular Probes, now part of Invitrogen, Eugene, OR) for 1 hour at 37uC under 5% CO 2 . The samples were mounted and viewed using the Zeiss Axioplan microscope described above, and live cells (bright green cytoplasm) and dead cells (bright blue nuclei) were enumerated in at least three fields per sample in at least three independent experiments. Second, the cells were stained using the Vybrant Apoptosis Assay Kit 2 (Molecular Probes), which stains for annexin V and membrane permeability. Third, the APO-BrdU TUNEL kit, also from Molecular Probes, was used. A second TUNEL-based kit, Alert DNA Fragmentation kit (Clontech Laboratories, Inc., Mountain View, CA) was also used. For methods two and three, the cells were also assessed using fluorescence microscopy. Finally, cells were harvested, and DNA was purified and analyzed for fragmentation (an assessment of apoptosis) using conventional agarose gel electrophoresis.