Figure 3.

Association and uptake of B. abortus-infected PMN by Mϕs. PMNs were incubated with B. abortus-RFP (red) (MOI of 50) for 1.5 h; then, cells were pelleted and washed with PBS to remove extracellular bacteria. Brucella-infected-PMNs were suspended in DMEM without gentamicin and added to RAW Mϕs monolayers (5 × 10³) at a rate of 1:1 and incubated for 10 min at 37°C. After this period, the infected Mϕ monolayers were washed and suspended in DMEM and incubated for up to 5 h. Infected PMNs were stained with Hoescht (blue). Cells were photographed and analyzed under Cytation 3 Cell Imaging Multi-Mode Reader (BioTek) using the appropriate color filter channel. Numbers 1 to 4 correspond to the order in the which images were capture very every 20 min. These experiments were repeated at least three times.