To investigate the reactive ROS level in mitochondria, mitochondrial extract was reacted with a respiration buffer including 125 mM KCl, 2 mM KH2SO4, 2.5 mM malate, 20 mM HEPES, 1 mM MgCl2, 5 mM pyruvate, 500 μM EGTA, and 25 μM DCF-DA. Then, the reactant was incubated in a dark room for 20 min, and fluorescence was measured using a fluorometer (Infinite F200, Tecan) at 485 nm (excitation wavelength) and 535 nm (emission wavelength) [29 (link)].
To measure the mitochondrial MMP level, the mitochondrial extract was reacted with MI buffer with 5 mM pyruvate, 5 mM malate, and 1 μM JC-1, and gently shaken. Then, the reactant was incubated in a dark room for 20 min, and fluorescence was measured at 530 nm (excitation wavelength) and 590 nm (emission wavelength) using a fluorometer (Infinite F200, Tecan) [30 (link)].
The ATP level was measured using an ATP kit (Sigma-Aldrich Chemical Co.) according to the manufacturer’s protocol. The reactant was then measured with a luminescence meter (GloMax, Promega, USA).
To measure the mitochondrial MMP level, the mitochondrial extract was reacted with MI buffer with 5 mM pyruvate, 5 mM malate, and 1 μM JC-1, and gently shaken. Then, the reactant was incubated in a dark room for 20 min, and fluorescence was measured at 530 nm (excitation wavelength) and 590 nm (emission wavelength) using a fluorometer (Infinite F200, Tecan) [30 (link)].
The ATP level was measured using an ATP kit (Sigma-Aldrich Chemical Co.) according to the manufacturer’s protocol. The reactant was then measured with a luminescence meter (GloMax, Promega, USA).
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