Visualizing Legionella Infection Dynamics

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).