Chapter 2 │ Page 44 Figure 2: Schematic representation of the experimental workflow. Parental SC263 cells were virally transduced and puromycin selected to obtain a stable red-fluorescent SC263T counterpart. Freshly-thawed SC263 and SC263T cells accounted for the young cellular counterparts (PL), whereas minimally 10 additional culture passages were performed before cells were considered for the older counterparts (PH). Time in culture and passaging was kept identical for parental and transduced cells in both young and old counterparts, respectively, and all experiments were performed within 2 additional passages (Px+2). To further characterize the cellular state of the di erent cellular conditions, proliferation and intracellular ROS level analysis was performed on all groups. Sensitivity for NTP was evaluated under standard culturing conditions, and after exposure (4 hours) to ambient light. 2.3 Cell Survival Analysis Following NTP treatment and 24-hour incubation, 2µM of the fluorescent dye Hoechst (in 0.3% Tween, 62249, ThermoFisher Scientific) and 0.05µM of Incucyte® Cytotox Green (in DMSO, 4633, Sartorius) were added to the wells with the D300e digital dispenser (Tecan, Switzerland) for cell labeling and cell death monitoring, respectively. The working concentration for the Incucyte® Cytotox Green was previously optimized in our lab and does not induce dye cytotoxicity [30]. Brightfield and fluorescence whole-well imaging (2x) was performed with the Tecan Spark® Cyto (Green laser Ex. 461-530 nm, Blue laser Ex. 381-450 nm) after 30 minutes of incubation. All images per experiment were analyzed together using the built-in Spark ControlTM Image Analyzer[33]. Briefly, whole-well analysis was performed to ensure evaluation of the entire cell population. Focus o set (Both channels: 0mm),
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