Macrophage-Bacterial Cell Association Assay

Cell association assays were performed similar to previous work, with the following modifications (19 (link)). Immortalized macrophage cells derived from wild-type mice (BEI Resources #NR-9456) were maintained in DMEM medium with L-glutamine, 4.5 g/L glucose and sodium pyruvate (Corning) supplemented with 10% heat-inactivated fetal calf serum (Corning), 100 U/mL penicillin, and 100 µg/mL streptomycin in an atmosphere of 5% CO₂. Confluent cells (~3 × 10⁵ cells/well) in 24-well tissue culture dishes were washed with 1 mL of PBS, and then 1 mL of 3 × 10⁶ CFU/mL bacteria (MOI 10) in additive-free DMEM was added to each well. Samples were spun at 500 rpm (54 × g) for 5 min and then incubated at 37°C, 5% CO₂ for 2 h, followed by an incubation at 4°C for 1 h. Samples were washed three times with PBS and then lysed with 1 mL of 0.2% Triton-X100 in PBS for 5 min. Input and cell-associated bacterial counts were determined by serial dilution and CFU enumeration on LB agar.