4.6.2. Mitochondrial ROS Content

To assess the ROS content in the mitochondria, the isolated mitochondria were mixed with 25 μM DCF-DA in respiration buffer (125 mM KCl, 2 mM KH₂PO₄, 2.5 mM malate, 20 mM HEPES, 1 mM MgCl₂, 5 mM pyruvate, and 500 μM EGTA) and left in a dark room for 20 min. After incubation, the fluorescence intensity was measured (excitation wave: 485 nm, emission wave: 535 nm) [8].