Mitochondrial Respiration Assay

Two types of experiments are presented with isolated mitochondria. In the first, respiration by the mitochondria (5 µg/well) was sequentially measured in a coupled state with substrate present (basal respiration), followed by State 3 (phosphorylating respiration, in the presence of ADP and substrate), State 4 (non-phosphorylating or resting respiration) following conversion of ADP to ATP, State 4_o (induced with the addition of oligomycin), and then maximal uncoupler-stimulated respiration (State 3_u). This allows respiratory control ratios (RCR; State 3/State 4_o, or State 3_u/State 4_o) to be assessed [18] –[20] . Unless otherwise noted, the substrate was 10 mM succinate plus 2 µM rotenone. Injections were as follows: port A, 50 µl of 40 mM ADP (4 mM final); port B, 55 µl of 25 µg/ml oligomycin (2.5 µg/ml final); port C, 60 µl of 40 µM FCCP (4 µM final); and port D, 65 µl of 40 µM antimycin A (4 µM final). The second type of experiment examined sequential electron flow through different complexes of the electron transport chain. With the initial presence of 5 µg mitochondria per well, 10 mM pyruvate, 2 mM malate and 4 µM FCCP, injections were made as follows: port A, 50 µl of 20 µM rotenone (2 µM final); port B, 55 µl of 100 mM succinate (10 mM final); port C, 60 µl of 40 µM antimycin A (4 µM final); port D, 65 µl of 100 mM ascorbate plus 1 mM N,N,N′,N′-Tetramethyl-p-phenylenediamine (TMPD, 10 mM and 100 µM final, respectively). Typical mix and measurement cycle times for the assays are illustrated in Table 1 and are common to all experiments presented unless otherwise noted.