Quantification of Oxidative Stress

Intracellular ROS were detected using the oxidation-sensitive fluorogenic dyes CellROX green (Life Technologies, Grand Island, NY, USA) or 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (Carboxy-H2DCFDA; ThermoFisher Scientific). For quantitative assessment of oxidative stress using CellROX green, TM cells were cultured in 8-well Nunc Lab-Tek II chambered glass slides (ThermoFisher Scientific) to near confluency, serum-starved overnight, and subsequently treated as described above. CellROX green (5 µM) was added to the final 30 minutes of treatment. Dye-loaded cells were rinsed with ice cold PBS and fixed for 20 minutes at 23°C by immersion in 4% buffered (pH 7.4) paraformaldehyde. Fixed cells were rinsed, air dried, and mounted with Fluoroshield containing 4′,6′-diamidino-2 phenylindole (DAPI). Cells were imaged using a Leica TCS SPE confocal microscope (Leica MicroSystems, Buffalo Grove, IL, USA) and LAS-X imaging suite. All cells’ fields were imaged under identical confocal conditions using identical software settings. For quantitative assessment of oxidative stress using carboxy-H2DCFDA, TM cells were cultured in 96-well plates to near confluency, serum-starved overnight, and subsequently incubated with 10 µM carboxy-H2DCFDA at 37°C for 30 minutes, as we have previously described.³¹ Carboxy-H2DCFDA pre-loaded cells were washed to remove excess dye and then treated as described above. Dichlorofluorescein diacetate fluorescence was quantified using a Labtech FLUOstar Optima luminometer (BMG Labtech, Cary, NC, USA) at 488-nm excitation/520-nm emission wavelengths.