Listeria Infection of Macrophages

The recombinant Listeria strains have been previously described [24 (link)]. For in vitro infections, they were grown to an OD₆₀₀ of 0.6–1.0 in BHI media (Sigma Aldrich) with 10 ug/ml chloramphenicol (Sigma Aldrich) at 30°C. Macrophages were infected with the Listeria strains using a MOI 50, for 45 minutes. Extracellular bacteria were eliminated by adding 60 ug/ml gentamicin (Sigma Aldrich) for 20 minutes. Bacterial burden was assessed by lysing the infected macrophages with 1% TritonX-100 in PBS, and plating serial dilutions of the lysate on BHI agarose supplemented with 10ug/ml chloramphenicol (Sigma Aldrich, St. Louis, MO). Recombinant listeriolysin (Prospec, East Brunswick, NJ) was added in some experiments at 2 ug/ml for 30 minutes, and any excess was washed away. Bafilomycin (InvivoGen, San Diego, CA) was added in some experiments at 5 uM for 30 minute, before being washed away.