Fig. 6. Confocal fluorescence images of live HEK293T cells in presence of chelators and a ROS sensitive dye. (a) Left to right: control cells; cells treated with chelator 2c (40 μM) for 30 min; with CuCl₂ (20 μM) for 30 min; with CuCl₂ (20 μM) for 30 min followed by 2c (40 μM) for 30 min. (b) Left to right: control cells; cells treated with 3c (40 μM) for 30 min; CuCl₂ (20 μM) for 30 min; CuCl₂ (20 μM) for 30 min followed by 3c (40 μM) for 30 min. Cells were stained with CellROX (5 μM) for 30 min at the final step before imaging. (λ_ex/em: 633/641–700 nm). Cells were washed three times with PBS buffer after each addition and then imaged. Lower panels of (a) and (b) shows bright field images of cells overlaid with confocal images. Fluorescence intensity analyses of confocal images were carried out by using ImageJ software. Bar plots represent the intensity analyses results for (c) chelator 2c and (d) chelator 3c. Intensity data were normalized to intensity of CuCl₂ treated cells. Error bars denote SEM, n = 3. Statistical analyses were performed using an unpaired, two-tailed Student's t-test (****p ≤ 0.0001). Images were acquired using a 40× objective; scale bar: 20 μm.