2.2 Culture Medium
and Extraction Buffer
- Culture medium for primary hepatocytes: M199 medium con-
taining 10% (v/v) FCS, 0.1% (w/v) BSA-FFA-free, 1 nM insu-
lin, 100 nM dexamethasone, 100 nM T3, and 100 IU/ml
penicillin and streptavidin. - Mitochondrial homogenization buffer: 250 mM sucrose,
20 mM Tris–HCl, pH 7.4, 1 mM EGTA. Add ~1.5 L of mQ
water to a glass beaker containing a magnet. Weigh 171.2 g of
sucrose and transfer to the glass beaker container. Add 4.8 g of
Tris-Base powder and 760 mg of ethylene glycol-bis (2-ami-
noethylether)-N,N,N^0 ,N^0 -tetraacetic acid (EGTA). Mix and
adjust pH to 7.4 with hydrochloric acid. Store at 4C over-
night and readjust pH to 7.4. Make up to 2 L with mQ water
and transfer in glass bottle. Store at 4C (weeks) or 20 C
(months).
2.3 Respiration
Buffers
- HEPES solution: 1 M 4-(2-hydroxyethyl)piperazine-1-ethane-
sulfonic acid, N-(2-hydroxyethyl)piperazine-N-
(^0) -(2-ethanesulfonic acid) (HEPES), pH 7.4. Add ~85 ml of
mQ water to a glass beaker containing a magnet. Weigh
23.83 g of HEPES. Mix and adjust pH to 7.4 with 10 M
sodium hydroxide. Make up to 100 ml with mQ water, filter
(0.22μM), and store at 4C.
- Krebs-HEPES-glucose buffer: 140 mM NaCl, 4.8 mM KCl,
1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 , 1.3 mM CaCl 2 ,11mM
glucose, 10 mM HEPES, pH 7.4 (seeNote 2). Add ~350 ml of
mQ water to a glass beaker containing a magnet. Weigh and
add 4.09 g of natrium chloride, 178.9 mg of potassium chlo-
ride, 81.7 mg of potassium phosphate monobasic, 72.2 mg of
magnesium sulfate, 72.1 mg of calcium chloride, 0.99 g ofD-
glucose. Add 5 ml of HEPES 1 M, pH 7.4. Mix and adjust pH
to 7.4 if necessary. Make up to 500 ml with mQ water and store
at 4C (weeks) or 20 C (months). - BSA solution: 15% (w/v) bovine serum albumin (BSA).
Add ~50 ml of mQ water to a glass beaker containing a mag-
net. Weigh 15 g of BSA [Fraction V, fatty acids-free] and
transfer to the glass beaker container. Mix and make up to
100 ml with mQ water. Aliquot in 15-ml Falcon tubes and
store at 20 C. - Pi-Tris: 1 M inorganic phosphate (Pi). Add 100 ml of mQ
water to a glass beaker containing a magnet. Add 13.6 ml of
phosphoric acid 85%. Mix and adjust pH to 7.2 with Tris-Base
powder. Make up to 200 ml with mQ water. Aliquot in 15-ml
Falcon tubes and store at 20 C. - KCl buffer: 125 mM KCl, 20 mM Tris, 1 mM EGTA, pH 7.2.
Add ~350 ml of mQ water to a glass beaker containing a
magnet. Weigh 4.65 g of KCl and transfer to the glass beaker
276 Guillaume Vial and Bruno Guigas