4.1.4. Intracellular ROS Levels

The ROS levels were measured using 2 ,7 -dichlorofluorescein diacetate (DCF-DA) (Sigma-Aldrich). Cells were plated at 2 × <sup>10</sup><sup>4</sup> cells/well in 96-well plates and incubated with test compounds at 37 ◦C and 5% CO2 for 24 h. Subsequently, the cells were washed three times with phosphate-buffered saline (PBS) and incubated in the dark with 20 μM DCF-DA for 30 min at 37 ◦C. The cells were washed twice with PBS. The fluorescence was measured using a fluorometer at 485 nm excitation/535 nm emission.

#### 4.1.5. Measurement of Mitochondrial Membrane Potential

Mitochondrial membrane potential was measured using tetramethylrhodamine (TMRE; Thermo Fisher Scientific, Scotts Valley, CA, USA). Cells were incubated with 200 nM TMRE in the dark for 30 min at 37 ◦C and 5% CO2. After washing, the fluorescence was measured at 550 nm excitation/590 nm emission and cells were imaged using a fluorescence microscope (Olympus, IX71; Olympus, Tokyo, Japan).
