ROS level. Isolation of mitochondria from brain tissues was performed as described previously [26 (link)]. The ROS production in mitochondria was determined using a respiration buffer (125 mM potassium chloride, 20 mM HEPES, 2.5 mM malate, 2 mM potassium phosphate, 5 mM pyruvate, 1 mM magnesium chloride and 500 μM EGTA, pH 7.0). A mitochondrial extraction was incubated with 25 μM DCF-DA in respiration buffer at room temperature for 20 min. Fluorescence was measured at a wavelength of 530 nm (excitation) and 590 nm (emission)(Infinite 200, Tecan Co., USA).
Mitochondrial membrane potential. mitochondrial membrane potential was conducted using the isolated mitochondria with a 200 μM JC-1. The assay buffer contained mitochondrial isolation buffer with 5 mM pyruvate and 5 mM malate. The assay buffer and mitochondrial extraction were added to the wells of a 96-well black plate, followed by the addition of 1 μM JC-1 and mixed gently. The microplate was covered with aluminum foil and left at room temperature for 20 min. Fluorescence (excitation wavelength: 530 nm, emission wavelength: 590 nm) was then measured (Infinite 200, Tecan Co.).
ATP Content. The previously preprocessed sample was centrifuged at 13,000 ×g for 10 min. The ATP level was measured using a commercial kit (Promega, USA) with a luminescence meter (GloMax-Multi, Promega).
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