Characterization of cell death induced by CB and TiO
NPs in 16HBE14o- cells. A) A dose response of FDA (fluorescein diacetate) and EtBr (ethidium bromide) staining after 4 hours of NP exposure (10-40 μg.cm-2) analyzed by fluorescence microscopy. 1 mM H2O2was used as positive control. B) Flow cytometry analysis of time course (0-24 h) of DNA fragmentation (% of cells in Sub-G1 peak). Cells were treated for the indicated durations with CB and TiO2 NPs (20 μg.cm-2), fixed and stained with propidium iodide for cell cycle analysis by flow cytometry. C) A time course study (0-24h) of the decrease in relative average cell size by measuring decrease in forward scatter (FS, % of control) of the laser in flow cytometry after NP exposures (20 μg.cm-2). D) Transmission electron microscopic analysis of ultrastructure. Cells were exposed for 24 hours to media alone (control) or NPs of CB and TiO2 and analyzed by TEM (control ×28000, CB ×28000 and TiO2 ×27000). Stars represent peripheral chromatin condensation, white arrow particle aggregates inside vesicles and grey arrow membrane blebbing. E) Time course analysis (0.5-24 h) of caspase-8 and caspase-3,-7 activation in CB and TiO2 NP treated cells (20 μg.cm-2). Cells were incubated with specific substrates for the respective caspases (Vybrant kit) and the % of caspase active cells was detected by flow cytometry. Data are represented as mean ± SD (n = 3). Representative results of at least three independent experiments. * statistically different from control p < 0.05 (two tailed).