Figure 2. Creation and validation of an in situ mitochondrial preparation.

A, optimizing saponin concentrations for respiration stimulated with glutamate, malate, and ADP or (B) succinate, rotenone, and ADP. C, verification of maintained membrane integrity performed through the addition of cytochrome c following succinate-stimulated respiration at several concentrations of saponin. D, confirmation of mitochondrial-stimulated respiration through complementing glutamate, malate, and ADP with rotenone, myxothiazole, or oligomyocin as complex I-, III- and V-specific inhibitors, respectively. Succinate-stimulated complex II respiration was performed in the presence of rotenone + ADP and inhibited with oxaloacetate. Maximal mitochondrial respiration was stimulated with serial additions of pyruvate, malate, ADP, glutamate, and succinate in succession. Pyruvate (E) and glutamate (F) titrations demonstrate a system responsive to changes in substrate availability and confirm saturating substrate concentrations for respiration. In both isolated mitochondria (G) and permeabilized brain (H), substrate responses to maximum respiration initially stimulated by pyruvate (white bars), glutamate (dark bars) or succinate alone (grey bars) show substrate-specific differences between mitochondrial preparations. n = 3–5 for all experiments. D, ADP; G, glutamate; M, malate; myx, myxothiazole; oligo, oligomyocin; oxalo, oxaloacetate; P, pyruvate; rot, rotenone; S, succinate.