- Add 20μl of ADP (stock: 100 mM; final concentration: 1 mM)
and measure theJO 2 coupled to oxidative phosphorylation
(state 3) during 1–2 min until reaching a steady state. - Add 1.5μl of oligomycin (stock: 1 mg/ml; final concentration:
0.75μg/ml), and measureJO 2 during 1–2 min until reaching a
steady state. The oligomycin-insensitiveJO 2 corresponds to
the proton leak though the mitochondrial inner membrane
(state 4). - Add 2μl of the OXPHOS uncoupler CCCP (intermediate
stock solution of 0.5 mM; final concentration: 0.5μM), and
measureJO 2 during 1–2 min until reaching a steady state in
order to assess the maximal mitochondrial respiratory rate in
uncoupled state. - Add 3μl of antimycin (stock: 100μg/ml; final concentration:
0.15μg/ml). MeasureJO 2 during 1–2 min until reaching a
steady state in order to assess the non-mitochondrial
respiration. - Add 50 μl of the respiratory-chain complex IV substrates
TMPD/ascorbate (stock: 20 mM/1 mM; final concentration:
0.5 mM/0.025 mM). MeasureJO 2 during 1–2 min until
reaching a steady state in order to assess the maximal activity
of cytochrome oxidase (respiratory-chain complex IV).
Fig. 4Methods for studying mitochondrial bioenergetics in permeabilized cells or isolated organelles. The
oxygen consumption rates (JO 2 ) of permeabilized cells (in situ mitochondria) or isolated mitochondria are
determined by measuring the decay of oxygen concentration in time (slope) in various respiratory states (a,b).
The basalJO 2 (pseudo-state 4) is measured in the presence of either glutamate/malate (oxidized at
respiratory-chain complex I), succinate (oxidized at respiratory-chain complex 2) plus rotenone (to inhibit
the reverse electron flow though the respiratory-chain complex I), or palmitoyl carnitine (oxidized by acyl-CoA
dehydrogenase). The subsequent step-by-step additions of ADP, oligomycin, CCCP, antimycin, and TMPD/
ascorbate (oxidized at respiratory-chain complex IV) allow to assess the respiration coupled to ATP synthesis
(state 3), the proton leak (state 4), the maximal respiratory rate in uncoupled state, the non-mitochondrial
respiration, and the maximal activity of cytochrome oxidase (respiratory-chain complex IV), respectively.
Adapted in part from [19]
282 Guillaume Vial and Bruno Guigas