A549 human alveolar epithelial cells were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum, 100 mg/liter of penicillin, and 100 mg/liter streptomycin at 37°C in the presence of 10% CO2. For cell viability assays, monolayers of A549 cells were cultured in 24-well plates to a density of 1 × 105 cells per well. Later, the cells were infected with 2 × 107 CFU/well of A. baumannii in new DMEM without antibiotics and grown for 20 h at 37°C (28, 29). The number of inoculated bacteria was determined by direct plating. A LIVE/DEAD fluorescence microscopy kit (Cellstain double-staining kit; Fluka, Buchs, Switzerland) was used according to the manufacturer's instructions to measure cell viability postinfection. Briefly, at 20 h postinfection, the A549 cells were incubated for 15 min at 37°C with phosphate-buffered saline (PBS) containing a mixture of the two fluorescent molecules to obtain simultaneous fluorescent staining; calcein-AM, is able to stain viable cells (green), while propidium iodide, a nucleus-staining dye that is unable to penetrate the cell membranes of viable cells, can stain only dead cells (red). Microscopic images of the stained cells (alive and dead) were obtained using an inverted fluorescence microscope (Nikon Eclipse Ti) and analyzed with the NIS Elements Br software package. The excitation absorbance of calcein-AM was 490 nm, and emission was at 515 nm, while the excitation range of propidium iodide was 535 nm and emission was 617 nm. At least six replicates of each assay were analyzed, and the statistical significance was determined using Student's t test. Full paper
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