Mitochondrial Function Assays

To evaluate the ROS level, mitochondrial extracts were reacted with respiration buffer (including 123 mM KCl, 2 mM KH₂PO₄, 2.5 mM malate, 20 mM HEPES, 1 mM MgCl₂, 5 mM pyruvate, and 500 μM EGTA) and 25 μM 2′,7′-dichlorofluorescin diacetate (DCF-DA). Then, the reactants were incubated in the dark for 20 min at 4 °C and fluorescence was measured at 485 nm (excitation wavelength) and 535 nm (emission wavelength) using a fluorescence microplate reader (Infinite F200, Tecan) [66 (link)].
To measure the MMP level, the mitochondrial extract was reacted with MI buffer with 5 mM pyruvate, 5 mM malate, and 1 μM JC-1. The reaction was gently mixed and incubated in the dark for 20 min at 4 °C. Then, fluorescence was measured at 530 nm (excitation wavelength) and 590 nm (emission wavelength) using a fluorescence microplate reader (Infinite F200, Tecan) [13 (link)].
To measure the ATP level, the mitochondrial extract was evaluated using a commercial ATP kit (Sigma-Aldrich chemical Co., Milwaukee, WI, USA). The ATP level was assessed according to the manufacturer’s protocol and reactants were measured using a luminometer (Glomax^®, Promega, Sunnyvale, CA, USA).