Heat inactivated fetal calf serum

A549 cells (ATCC CCL185) derived from a human lung carcinoma were maintained in Ham’s F-12K (Kaighn’s) medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum (Corning), 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO₂.
Bacterial pellets from stationary phase cultures were resuspended in 1 mL F-12K without additives then normalized to 2x10⁷ CFU/ml (OD 0.02). Confluent A549 cells (~5x10⁵ cells/well) in 24-well tissue culture dishes were washed with 1 mL of PBS then 1 mL of 2x10⁷ CFU/ml bacteria (MOI 50) in additive-free F-12K were added to each well. Samples were spun at 500 rpm (54 x g) for 5 min then incubated at 37°C, 5% CO₂ for 1 h, followed by an incubation at 4°C for 1 h. Samples were washed three times with PBS then lysed with 1 mL of 0.2% Triton-X100 in PBS for 5–10 min. Input and cell-associated bacterial counts were determined by serial dilution and CFU enumeration.

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.