- CCCP: 100 mM carbonyl cyanide 3-chlorophenylhydrazone
(CCCP). Dissolve 20.5 mg of CCCP in 1 ml of DMSO.
Aliquot (100μl) in 0.5 ml tubes and store at 20 C. - DNP: 100 mM 2,4-dinitrophenol (DNP). Dissolve 18.4 mg of
DNP in 1 ml of DMSO. Aliquot (100μl) in 0.5 ml tubes and
store at 20 C. - Antimycin: 100μg/ml antimycin. Dissolve 5 mg of antimycin
A in 50 ml of ethanol absolute. Aliquot (250μl) in 0.5 ml tubes
and store at 20 C. - Myxothiazol: 1.6 mM myxothiazol: Dissolve 3.9 mg of myx-
othiazol in 5 ml of DMSO. Aliquot (100μl) in 0.5 ml tubes
and store at 20 C. - Ascorbate: 0.5 M ascorbate. Dissolve 0.88 g ofL-ascorbic acid
in 10 ml of Tris-buffer 50 mM. Aliquot (250μl) in 0.5 ml tubes
and store at 20 C. - TMPD/ascorbate: 20 mM N,N,N^0 ,N^0 -tetramethyl-p-phenyle-
nediamine dihydrochloride (TMPD), 1 mM ascorbate. Add
47.4 mg of TMPD and 20μl of ascorbate 0.5 M in 10 ml of
warm mQ water or medium. Aliquot (250μl) in 0.5 ml tubes
and store at 20 C. - Digitonin solution: 10% digitonin. Dissolve 100 mg of digito-
nin in 1 ml of DMSO. Aliquot (100μl) in 0.5 ml tubes and
store at 20 C. - Glucose: 1 mM. Dissolve 1.8 g ofD-glucose in 10 ml of mQ
water. Aliquot (250μl) in 0.5 ml tubes and store at 20 C.
3 Methods
3.1 Determination
of Mitochondrial
Oxygen Consumption
Rates in Intact Cells
Experiments are usually carried out at 37C.3.1.1 Clark-Type Oxygen
Electrode
- Calibrate the Clark-type polarographic oxygen electrode device
according to the manufacturer’s guidelines (seeNote 3). - Preincubate freshly isolated primary hepatocytes (~2.10^7 cells;
seeNotes 4and 5 ) for ~30 min at 37C in a shaking water bath
in closed vials containing 2 ml of Krebs-HEPES-glucose buffer
with or without additional exogenous substrates (seeNote 6). - Saturate the cell suspension or not with a gas phase containing
O 2 /CO 2 (19:1) for ~1 min, depending of the sensitivity of the
Clark electrode used. - Transfer the cells into the oxygraph chamber.
278 Guillaume Vial and Bruno Guigas