24 well tissue culture plate

A human intestinal epithelium cell line (INT-407) was cultured at 37 °C under 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM; HyClone Laboratories Inc., Logan, UT, USA) containing 10% fetal bovine serum (FBS) and 100 mg/L gentamicin. The cultured cells were seeded (10⁶ cells/mL) into 24-well tissue culture plates (BD Falcon, Franklin Lakes, NJ, USA) to reach >90% confluence at 37 °C. The post-confluent cultures were rinsed twice with PBS and preincubated in prewarmed antibiotic-free DMEM for 2 h. After stabilization, the INT-407 cell monolayers were used to evaluate the invasive ability of ST and SE cells exposed to different bile conditions. ST and SE cells exposed to 0%, 0.1%, or 1% bile were suspended in antibiotic-free DMEM (approximately 8.0 × 10⁵ CFU/mL) and infected into the INT-407 cell monolayers at 37 °C for 2 h. After infection, the INT-407 cell monolayers were incubated at 37 °C for 2 h in DMEM containing 100 mg/L gentamicin to inactivate extracellular ST and SE cells, rinsed with PBS to eliminate the gentamicin residue, and lysed with 1% Triton X-100 for 15 min at 37 °C. The lysates were serially diluted (1:10) with PBS. The proper dilutions were plated on LB-agar and incubated at 37 °C for 24–48 h, and intracellular ST and SE cells were counted^{36 (link)}. Each invasion assay was performed at least three times (four wells per assay).

The A549 cell line (American Type Culture Collection) is a human alveolar epithelial carcinoma cell line. A549 cells were grown in F-12 K tissue culture medium supplemented with 10% fetal bovine serum and penicillin/streptomycin 100 U/ml (Gibco) in a humidified atmosphere at 37 °C with 5% CO₂. For bacterial invasion and replication experiments, A549 cells (2 × 10⁵ cells per well) were seeded in 24-well tissue culture plates (BD Falcon). The cultures were grown at 37 °C with 5% CO₂. Prior to infection, cells were incubated overnight in antibiotic-free medium.

A549 and FaDu epithelial cell monolayers were seeded in 24-well tissue culture plates (Falcon – Corning, New York, USA) at a density of 5 × 10⁵ cells per well on the day before the experiments. Cells were infected with biofilm-dispersed and planktonically growing Kp-Sp^R such that the multiplicity of infection was 1 bacterium per cell (MOI₁). To determine bacterial adhesion, chloramphenicol at 35 µg/mL was added to the culture medium to inhibit bacterial growth. After 3 h of incubation, cells were washed two times with PBS, and lysed with 1% Triton X-100 in PBS. Samples were serially diluted, plated onto LB agar plates, and CFUs were determined.

FDC-P1 cells transfected with TAP-Lxn and TAP control vectors were treated with (1) 3 Gy or 6.5 Gy γ-irradiation or (2) VP-16 (Etoposide, Sigma-Aldrich) at the concentration of 20 or 30 μg/ml for 1 h. In each treatment group, 5 × 10⁴ cells were seeded into 24-well tissue culture plates (BD Falcon, Franklin Lakes, NJ, USA) and at day 0. The cell numbers were subsequently counted on a hemacytometer using trypan blue dye exclusion at different time points for 9 days. Fresh medium was added at each time point. FACS analysis was performed at each time point to measure the percentage of GFP+ cells.