Detection of Reactive Oxygen Species (ROS). Figure 3A: Detection of ROS using DHE fluorescent dye in CHME-5 and SH-SY5Y cells from the two treatment groups (DMSO control and 5 nm rotenone) at the end of week 4. Hoechst 33258 stain (blue) was used to identify the nucleus of cells. The fluorescent intensity of the red dots in the nucleus indicates the relative levels of ROS present in the cell. Scale bar: 20 μm. Figure 3B: Histogram showing the levels of ROS in each of the treatment groups in both cell types (CHME-5 and SH-SY5Y) at the end of week 4. The levels are expressed in densitometric units. P (*p > 0.05) value indicates that there was no significant difference in ROS levels between the control and rotenone treated CHME-5 cells. However rotenone treated SH-SY5Y cells showed a significant (**P < 0.001) two fold increase in the intracellular ROS levels compared to the untreated controls. Each column represents the mean and standard deviation from three independent experiments Figure 3C: Histogram representing the extracellular levels of ROS using the Acridan Lumigen PS-3 assay in the medium collected from both treatment groups (DMSO control and 5 nm rotenone) and both cell types (CHME-5 and SH-SY5Y) at the end of week 4. The levels are expressed in light intensity units. CHME-5 cells released significantly higher (*p < 0.001) levels of ROS in the medium compared to controls, while the extracellular release of ROS in the medium from rotenone treated and untreated SH-SY5Y cells was not significantly different (**P > 0.05). Each column represents the mean and standard deviation from three independent experiments. Figure 3D: Image showing the effects of antioxidants on chemiluminescence in the Acridan Lumigen PS-3 assay. Chemiluminescent signals were captured using Fujifilm Luminescent Image Analyzer (Dots) and light intensity units were recorded using the BioTek Synergy 2 plate reader (bar graph). a: culture medium from CHME-5 cells treated with 5 nm rotenone for 4 weeks, b: culture medium (as in a) incubated with 100 μM ascorbic acid (vitamin C) for 5 minutes, c: culture medium (as in a) incubated with NuPAGE antioxidant (1:500 diluted). The dramatic reduction of chemiluminescence in the media incubated with antioxidants indicates that the chemiluminescence in Acridan PS-3 assay is ROS specific.