Figure 4.

(a) Showing ACH induced oxidative stress in TR146 oral keratinocytes. Cells were incubated with ACH (25 µM – 100 µM) in 96 well plates. 30 mM H₂O₂ was used as positive control and medium containing PBS as a negative control. Reactive oxygen species (ROS) were detected by measuring DCF-DA fluorescence in a microplate reader (Genios, Tecan) in the presence and absence of N-acetylcysteine (NAC). Results are the average of three separate experiments. (b) Showing fluorescence in TR146 cells stained with DCF-DA to detect ROS (green) and Hoechst to detect nuclei (blue). Cells were preincubated in 100 µM of DCF-DA with or without NAC. Cells were then exposed to stress (50 or 100 µM ACH or 30 mM H₂O₂) and visualized using a Zeiss epifluorescence microscope