- Start the acquisition of the basal oxygen consumption rate
(JO 2 ) until reaching a steady state (usually ~1–2 min, Fig.2). - Add 3μl of the respiratory-chain ATP synthase inhibitor oli-
gomycin (stock: 1 mg/ml; final concentration: 1.5μg/ml)
with a Hamilton syringe through the cap of the oxygraph
chamber and measureJO 2 during 1–2 min until reaching a
steady state. The oligomycin-sensitiveJO 2 corresponds to the
mitochondrial respiration coupled to ATP synthesis and the
oligomycin-insensitive JO 2 to the proton leak though the
mitochondrial inner membrane. - Add 2μl of the OXPHOS uncoupler CCCP (prepare an inter-
mediate solution of 0.5 mM by mixing 10μl of the 100 mM
stock with 990μl of DMSO; final concentration: 0.5μM;see
Note 7) and measureJO 2 during 1–2 min until reaching a
steady state in order to assess the maximal mitochondrial respi-
ratory rate in uncoupled state. - Add 3μl of the respiratory-chain complex III inhibitor anti-
mycin (stock: 100μg/ml; final concentration: 0.15μg/ml;see
Note 8) and 2.5μl of the respiratory-chain complex I inhibitor
rotenone (stock: 2 mM; final concentration: 2.5μM). Measure
JO 2 during 1–2 min until reaching a steady state in order to
assess the non-mitochondrial respiration. - Wash carefully the oxygraph chamber after each measure with
mQ water (three times), then 70% ethanol (three times) and
again mQ water (three times), in order to remove any remain-
ing traces of inhibitors.
Fig. 2Methods for studying mitochondrial bioenergetics in intact cells. The oxygen consumption rates (JO 2 )of
primary or cultured intact cells are determined by measuring the decay of oxygen concentration in time (slope)
in various respiratory states (a,b). The basalJO 2 can be measured in the absence (endogenous) or presence
of various exogenous substrates (glucose, fatty acids, etc.). The subsequent step-by-step additions of
oligomycin (respiratory-chain ATP synthase inhibitor), CCCP (OXPHOS uncoupler), and antimycin
(respiratory-chain complex III inhibitor) plus rotenone (respiratory-chain complex I inhibitor) allow to assess
the mitochondrial respiration coupled to ATP synthesis (oligomycin-sensitive JO 2 ), the proton leak
(oligomycin-insensitiveJO 2 ), the maximal respiratory rate in uncoupled state, and the non-mitochondrial
respiration (antimycin/rotenone-insensitiveJO^2 ), respectively. Adapted in part from [19]
Methods for Assessing Mitochondrial OXPHOS 279