Fig. 2. Schematic of standard experimental protocol to investigate the contribution of components of the mitochondrial respiratory chain to the oxygen (O₂) consumption rate (OCR).

a Mitochondrial function can be thoroughly investigated in intact cells by measuring the OCR during sequential addition of mitochondrial respiratory inhibitors (marked with grey triangles). The different stages of the experiment (i)–(iv) and the measured parameters (a)–(f) are described in Protocol 1. The addition of pharmacological compounds or fuel substrates prior to oligomycin (not shown here) can capture further detail regarding the OCR. b Illustration of the effects of relevant pharmacological compounds on the mitochondrial respiratory chain, proton (H⁺) leak across the mitochondrial inner membrane, and the F₁F_o ATP synthase, during the experimental stages marked as (i)–(iv) in A. O₂ in the mitochondria is consumed by the respiratory chain through the activity of Complex IV. (i) In the basal state, mitochondrial O₂ consumption is predominantly driven by H⁺ flux through the F₁F_o ATP synthase. (ii) Inhibition of the F₁F_o ATP synthase with oligomycin reduces mitochondrial O₂ consumption, with the OCR in this phase predominantly driven by the H⁺ leak (but also by substrate oxidation). (iii) Addition of an uncoupler such as FCCP or CCCP increases the H⁺ leak across the inner membrane, creating a H⁺ short circuit and facilitating the measurement of maximal OCR. The optimal FCCP/CCCP concentration to induce maximal respiration should be determined for each experimental setting (details in Protocol 1), and it is advisable to also assess maximal respiratory capacity in the absence of oligomycin. (iv) Inhibition of respiratory chain activity with Rotenone and/or Antimycin A ablates mitochondrial O₂ consumption. Any O₂ consumption measured in this phase is due to non-mitochondrial O₂ consumption. Rot rotenone, AA antimycin A, Oligo oligomycin, H⁺ proton, IMS intermembrane space. *Respiration in stage (b) is predominantly driven by H⁺ leak, but also by substrate oxidation