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Respiratory control can be investigated outside an intact biological cell by supplying isolated mitochondria with substrates and ADP. This makes the boundary between oxygen consumption and ATP formation experimentally accessible. The historical mitochondrial oxygen electrode assay describes the 1955 experiment; the following authored protocol adds modern preparation and inhibitor controls.

In Functional Assessment of Isolated Mitochondria In Vitro (2009), Ian R. Lanza and K. Sreekumaran Nair describe human skeletal-muscle biopsy mincing, cold protease treatment, homogenization and differential centrifugation. A low-speed spin removes coarse material; subsequent spins collect and wash mitochondria. A stirred, closed $2\mathrm{mL}$ Oxygraph-2k chamber at $37{}^{\circ }\mathrm{C}$ uses a calibrated polarographic oxygen electrode. Its signal gives dissolved oxygen concentration; the negative time derivative gives consumption. Instrument corrections include electrode consumption and oxygen leakage.^[ref]

Their additions include glutamate plus malate, an ADP pulse, then saturating ADP. Added cytochrome c tests outer-membrane damage: stimulation suggests loss of endogenous cytochrome c. Later oligomycin inhibits ATP synthase; FCCP produces respiratory uncoupling; antimycin A inhibits respiratory complex III to estimate residual background. Figure 6 includes intervening succinate dehydrogenase and respiratory complex I interventions; the modeled traces below simplify that sequence. Isolation can damage or select mitochondria, and saturating assay conditions differ from those in living biological tissue.^[ref]

The control logic matters more than a single steep slope. If ADP accelerates consumption and a finite pulse is followed by slowing, demand is affecting respiratory flux. If oligomycin slows consumption, but subsequent FCCP accelerates it, the electron transport chain can still turn over while ATP synthase remains inhibited. That contrast separates restricted coupled turnover from a completely inactive chain. Failure of FCCP to restore a rate is less specific: substrate limitation, damage, low oxygen or excessive uncoupler can also restrict respiration.

Neither the cytochrome c test alone nor a large respiratory control ratio establishes every aspect of mitochondrial integrity. Likewise, oxygen consumption is not a direct ATP assay: respiratory uncoupling provides a concrete counterexample. The preparation, additions and normalization denominator determine what comparison the experiment can support.

The respiratory control ratio divides the oxygen consumption rate during ADP-stimulated respiration by that after supplied ADP has been used. It compares reaction rates, not ATP formed per oxygen.^[ref]

A large ratio means strong stimulation under the comparison's conditions; it is not a universal measure of all mitochondrial functions.

For a constructed example, choose constant suspension volume $V=2\mathrm{mL}$ and oxygen concentration declines of $0.030$ and $0.006\mathrm{mmol}{\mathrm{L}}^{-1}\mathrm{mi}{\mathrm{n}}^{-1}$ during the two comparison windows. Assume no gas exchange or dilution and a correctly calibrated readout. A derivative converts each concentration slope into an amount of substance consumption rate:The common volume cancels in the ratio, so dividing the positive slope magnitudes gives the same answer. These are chosen inputs, not the 1955 observations. If an uncorrected influx of $3\mathrm{nmol}\mathrm{mi}{\mathrm{n}}^{-1}$ of oxygen occurred in both windows, the apparent rates would instead be $57$ and $9$, giving $19/3\approx 6.33$. The same absolute error can inflate a ratio because it removes a larger fraction of its smaller denominator.