Bacterial Strains. Where Broth Grown Bacteria Were Used for Infections, Strains Were Cultured

Bacterial strains. Where broth grown bacteria were used for infections, strains were cultured in ACES-buffered yeast extract media overnight to an OD600 range of 3.5-5.2. Cultures within this optical density range containing motile bacteria (judged visually) were used for infection. To induce expression of dsRed, L. pneumophila strains were grown on plates or in liquid media containing 0.5 mM IPTG.

Mice and bone-marrow derived macrophage cell culturing. Nlrc4-/-, Asc-/- and Casp1-/- mice were backcrossed to the C57BL/6 background for 5, 9 and 10 generations, respectively. C57BL/6 mice were purchased from Jackson Laboratories.Bone marrow was collected from the femurs and tibiae of mice and cultured for 7 days in RPMI 1640 containing 20% fetal bovine serum (FBS), 25% macrophage colony-stimulating factor (M-CSF) and penicillin/streptomycin (100 units/ml). M-CSF used in macrophage culture media was from supernatants of L-929 fibroblast cells (ATCC).

Microscopy. Cells were washed with PBS and fixed in 4% paraformaldehyde prior to immunofluorescent processing. Cells were permeabilized by briefly incubating coverslips in cold methanol followed by blocking in PBS containing 2% goat serum (or 2% BSA when goat-derived antibodies were used) and 50 mM NH4Cl for 20 minutes at 37oC. Primary and secondary antibodies were diluted in PBS containing 2% goat serum or BSA. Cells were incubated with primary antibodies for 60 minutes at room temperature followed by washes in PBS. Next, cells were incubated with secondary antibodies for 30 minutes at room temperature followed by incubation in PBS containing Hoechst 33342 (Invitrogen) at 2.5 g/ml for labeling of DNA. Coverslips were mounted in ProLongantifade reagent (Invitrogen) and imaged on a Nikon Eclipse TE2000-S microscope using a 100x/1.4 numerical aperture lens unless indicated otherwise. Images were acquired using a Hamamatsu ORCA-ER camera controlled by IP lab and analyzed using Image J (NIH).

Live-cell imaging was performed on retrovirally-transduced bone marrow-derived macrophages. Cells were platedin 35 mm dishes having cover glass bottoms (MatTek) and infected at an MOI of 5 with broth grown L. pneumophila expressing dsRed. Images were taken using a 60x/1.4 numerical aperture objective of a Volocity spinning-disc confocal microscope equipped with a LiveCell environmental chamber (Pathology Devices). Time-lapse videos were edited using the Volocity software package (PerkinElmer).