Alexa fluor 568 phalloidin

F- and G-actin were visualized from Legionella-infected macrophages using Alexa Fluor® 568 phalloidin and Alexa Fluor® 488 DNAse I (Molecular Probes) and for studies examining colocalization of GFP-expressing bacteria (Legionella and E. coli) with the lysosome, Lysotracker™ red was used to stain acidic vesicles of infected BMDMs, as previously described14 (link). To examine cell death, after infection macrophages were stained with 4 uM ethidium homodimer-1 (EthD-1) from the LIVE/DEAD^® viability/cytotoxicity kit (Molecular Probes), according to manufacturer’s instructions. Images were captured using laser scanning confocal fluorescence microscope with a 60X objective (Olympus Fluoview FV10i).

A549 cells were seeded on chamber slides coated with collagen I (5x10⁴/ well) and cultured for 1 day in absence of antibiotic, while Calu-3 cells were cultured at ALI for 10–14 days, as described previously.
A549 cells and ALI cultured Calu-3 cells were incubated with fluorescent bacteria as described in the previous paragraph. After washes, A549 cells were fixed with 3.7 % formaldehyde (Sigma) for 20 min and were then incubated with Alexa Fluor® 568 phalloidin (Molecular Probes, Life Technologies) diluted 1:400 in PBS + 0.1 % Tween20 + 1 % BSA for 30 min. Calu-3 cells were fixed directly on inserts by adding 3.7 % paraformaldehyde in the apical and basal chamber and were then permeabilized with 0.2 % Triton X-100 diluted in PBS for 5 min. Subsequently, samples were blocked with PBS containing 3 % Bovine Serum Albumin (BSA) (Sigma) for 15 min and incubated with rabbit ZO-1 polyclonal antibodies diluted in PBS + 1 % BSA (1:200) for 1 h. After washes, samples were incubated with Alexa Fluor® 568 goat anti-rabbit IgG (1:500) (Molecular Probes) for 30 min. After three washes with PBS, the insert was carefully detached by a scalpel and mounted on a microscope slide by adding one drop of ProLong® Gold Antifade Mountant with DAPI. Images were analyzed with Zeiss LSM710 confocal microscope.