*4.6. Mitochondrial Oxidative Stress*

Intracellular oxidative stress level was measured by <sup>3</sup>-(*p*-aminophenyl)-fluorescein (APF). Cells cultured in T25 flasks were washed with PBS and treated with 5 μM APF in PBS for 60 min at 37 ◦C in a 5% CO2 incubator. After the treatment, cells were trypsinized, suspended in PBS at 4 × 10<sup>4</sup> cells/mL, and green fluorescent intensity was measured by a fluorometer (JASCO, Tokyo, Japan). The excitation and emission wavelengths were set up at 490 nm and 515 nm, respectively.

Mitochondrial damage was quantified by MitoSox-Red. Cells cultured in T25 flasks were washed with PBS and treated with 1 μM MitoSox-Red (Invitrogen) in PBS for 20 min at 37 ◦C in a 5% CO2 incubator. After the treatment, cells were trypsinized, suspended in PBS at 4 × 10<sup>4</sup> cells/mL, and red fluorescent intensity was measured by a fluorometer (JASCO, Tokyo, Japan). The excitation and emission wavelengths were set up at 400 nm and 580 nm, respectively. The nuclei were counterstained with 0.1 mg/mL of DAPI. Relative fluorescence was calculated by dividing total green or red fluorescence by total blue fluorescence obtained by DAPI staining.
