Chapter 2 │ Page 46 ThermoFisher Scientific). This cell-permeant dye, upon oxidation, binds to DNA and exhibits photostable fluorescence with excitation and emission maxima at 485 and 520 nm, respectively. After 30 minutes of incubation, baseline intracellular ROS levels were obtained with the Incucyte ZOOM® imaging system (Green laser Ex./Em.: 441-481/503-544 nm) [34]. Fold change of mean green fluorescence (green calibrated unit; GCU) was calculated for both transduced and parental cells at low and high passages. For the assessment of ROS induction after repetitive fluorescent excitation, cells were prepared as described above. Phase contrast and fluorescence images were captured every 2 hours up to 48 hours in the Incucyte ZOOM® live-cell imaging system and the intracellular ROS level was measured with CellROXTM. Analysis was performed using the Incucyte ZOOM® software and mean GCU was measured following background subtraction and thresholding. All images per experiment were analyzed together using the same image analysis parameters to limit variability. 2.6 Statistical Analysis For kinetic, live-cell imaging analysis, statistical significance was determined using a Two-Way ANOVA. The mean values of the cell lines (confluence or GCU) were compared to each other at each specific time point. A Tukey test was performed to correct for multiple comparisons. For baseline intracellular ROS levels, fold change of the mean GCU was calculated and a One-Way ANOVA with a Dunn's multiple comparisons test was used to determine significance. For the analysis of intracellular ROS levels over time, statistical significance was determined using a mixed-e ect analysis. The mean GCU values of the cell lines were compared to each other at each time point and a Holm-Sidak’s multiple comparison test was performed. Data in the figures are presented as mean ± standard error of the mean (SEM).
RkJQdWJsaXNoZXIy MTk4NDMw