Rpmi 1600 medium

1 × 10⁶ PBMC were suspended in RPMI 1600 medium (Gibco, Life Technologies, USA) with 10 % FBS, 200 mM l-glutamine and 5 mg/ml gentamicin (Sigma–Aldrich, Saint Louis, MO, USA) and cultured with Leukocyte Activation Cocktail with BD Golgi Plug from BD Biosciences (San Jose, CA, USA) for 4 h at 37 °C in a 5 % CO₂ humidified atmosphere. After this time, cells were washed with PBS+ 1 %FBS and stained with monoclonal antibodies anti-CD3–PerCP (Clone SK7), anti-CD4–APC (Clone RPA-T4), anti-CD8–APC-H7 (Clone SK1) from BD Biosciences (San Jose, CA, USA). Next, cells were washed with PBS+ 1 %FBS and suspended in Fixation/Permeabilization Solution (BD Biosciences, San Diego, CA, USA). After permeabilization, cells were washed with Perm/Wash/Buffer (BD Biosciences, San Diego, CA, USA) and stained with monoclonal antibodies: anti-IFNγ–FITC (Clone B27), anti-IL-17A–PE (Clone N49-653), anti-TNF–FITC (Clone MAb11) and anti-IL-4–PE (Clone 8D4-8). In some cases, isotype controls: IgG₁ κ–FITC (Clone MOPC-21) for IFNγ and TNF, IgG₁κ–PE (Clone MOPC-21) for IL-17A and IL-4 were used (BD Biosciences, San Jose, CA, USA). All staining and permeabilization were performed for 20 min in the dark at 4 °C. After intracellular staining, cells were washed with Perm/Wash Buffer, suspended in PBS+ 1 %FBS and collected using a FACS Canto II flow cytometer from BD Biosciences (San Jose, CA, USA).

E. coli strain CFT073 and its derivative CFT073ΔcpxA::cat were described previously and maintained on Luria broth (LB) plates at 37°C (30 (link)). The E. coliPcpxP′-lacZ reporter strain, PAD282, was the gift of Thomas Silhavy (Princeton University, Princeton, NJ, USA) and was maintained on TB plates at 37°C (31 (link)). HepG2 hepatocellular carcinoma cells were the gift of Andy Yu (Indiana University, Indianapolis, IN) and were grown in RPMI 1600 medium (Gibco) containing 10% fetal calf serum (HyClone) and 1 mM sodium pyruvate (Sigma) at 37°C with 5% CO₂ as described (31 (link)).