L. monocytogenes-infected cells were stained as described, followed by incubation with a secondary Alexa 488-labeled anti-rabbit polyclonal antibody (Molecular Probes, 1:2,000 dilution), and F-actin was stained with tetramethylrhodamine B isothiocyanatephalloidin (Sigma, 1:1,000 dilution). Both primary and secondary antibodies as well as phalloidin were incubated at room temperature for 60 minutes followed by three washes with TBST buffer. Coverslips were mounted as described above. Bacterial escape was quantified by the number of actin associated (red, phalloidin-positive) fluorescent bacteria over the total number of intracellular green fluorescent bacteria at 30, 60, 90 and 120 minutes post-infection. The percentage of escape was determined by dividing the number of phalloidin-positive (escaped) bacteria by the number of total intracellular bacteria [58] . Numbers were obtained from the average of a minimum of three experiments.