Fig. 7. Simultaneous imaging of copper levels and oxidative stress in live HEK293T cells in the presence and absence of chelators 2c and 3c. Phen Green FL was used to image Cu ions and CellROX was used to image ROS. (A) Top to bottom (a–d): confocal images of control untreated cells; cells treated with CuCl2 (20 μM) for 30 min; cells treated with CuCl2 (20 μM) for 30 min followed by 2c (80 μM) for 30 min; cells treated with CuCl2 (20 μM) for 30 min followed by 3c (80 μM) for 30 min. All cells were incubated with Phen Green FL (5 μM) for 30 min prior to any other treatment (λex/em: 488/498–600 nm) and incubated with CellROX (5 μM) for 30 min at the final step before imaging (λex/em: 633/641–700 nm). Cells were washed three times with PBS buffer after each addition and then imaged. Number of washings was identical for all experiments. Left to right: HEK293T cells showing Phen Green FL emission, CellROX emission, and overlaid confocal images. (B) The white lines in the overlaid images were used to calculate the fluorescence intensity of Phen Green FL (green) and CellROX (blue) in the line scan along the direction of 1 to 2. (C) Bar plots represent the intensity analyses results for panel (A, a–d). Intensity data were normalized to intensity of control untreated or CuCl2 treated cells. Error bars denote SEM, n = 3. Images were acquired using a 40× objective; scale bar: 20 μm.
