Phalloidin 555

After bacterial infection, cells were washed with PBS and fixed in 3.7% paraformaldehyde for 10 minutes at room temperature. Cells were then washed three times with PBS and subsequently blocked with 5% BSA in PBS-T (containing 0.1% Triton X-100; Sigma-Aldrich, St. Louis, MO). For observation, cells were stained with 5 μg/mL phalloidin-555 (Molecular Probes, CA) and 1 μg/mL DAPI (Molecular Probes, CA). Alternatively, the BSA-blocked cells were stained with a mouse anti-αvβ3 primary antibody (1:100; ab7166, Abcam, Cambridge, UK) and a rabbit anti-Salmonella antibody (1:100; ab35156, Abcam, Cambridge, UK) overnight at 4°C. After washing three times with PBS-T, cells were incubated with anti-mouse IgG coupled to Alexa Fluor 555 (1:500; Molecular Probes) and anti-rabbit IgG coupled to Alexa 488 (1:500; Molecular Probes) for 2 h at room temperature. Stained cells were subsequently washed with PBS, mounted on glass slides, and analyzed under an FV1000D confocal laser scanning microscope (Olympus, Tokyo, Japan). For binding analysis, a composite image of 20 sections with a 0.5 μm shift in the z-axis was taken and combined using FV10-ASW 2.0 viewer. The number of adherent bacteria was determined by counting at least 100 high power fields (320 μm × 240 μm). Calculation of exact cell numbers was based on the measured areas and the overall size of the cover slip.