Evaluation of autophagic vacuole formation in A549 cells infected with NTHi strain. After infection for indicated times (2, 4, 6, 8, and 12 h), A549 cells were washed with PBS and incubated with fresh medium containing (a) AO (1 mg mL⁻¹) for 15 min and (b) 0.05 mM monodansylcadaverine in PBS for 10 min, washed with cold PBS and immediately examined by fluorescence microscopy. Uninfected cells were taken as control. Representative images were obtained from two independent experiments. (Inset: Enlarged microscopic view, 100×). (c) After incubation with 0.05 mM monodansylcadaverine at 37°C for 10 min, cells were washed with PBS and collected in 10 mM Tris-HCl, containing 0.1% Triton X-100. The amount of incorporated monodansylcadaverine was quantitated using Cary Eclipse fluorometer. The data represent the mean ± SEM of three independent experiments. ***P ≤ 0.001 indicates statistically significant difference between time points of infection.