- Port B (CCCP): prepare 2.4 ml of intermediate solution by
adding 36μl of CCCP 1:100 (stock: 100 mM; 1:100 stock:
1 mM; final concentration: 1.5 μM) in 2364 μlof0%
FCS/XF assay medium. - Port C (antimycinþrotenone): prepare 2.4 ml of interme-
diate solution by adding 132 μl of antimycin (stock:
100 μg/ml; final concentration: 0.5μg/ml) and 13.2μlof
rotenone (stock: 2 mM; final concentration: 1 μM) in
2255 μl of 0% FCS/XF assay medium.
- Replace the M199 complete medium by 180μl of 5% FCS/XF
assay medium. - Incubate the cells for 1 h at 37C in a dry incubator in order to
remove the CO 2 dissolved in the XF assay medium. - Set up the assay design (number of measurements, timing of
injection, etc.) using the Seahorse XF Wave software. - Start the automated calibration of the system.
- Load the 96-well plate and start the acquisition (seeNote 10).
3.2 Determination
of Mitochondrial
Oxygen Consumption
Rates in Permeabilized
Cells
The mitochondrial bioenergetics can be assessed in permeabilized
hepatocytes (in situ organelles) using Clark-type polarographic
oxygen electrode devices (Fig.1b). Experiments are usually carried
out at 37C.- Calibrate the Clark-type polarographic oxygen electrode, as
described in Subheading3.1.1. - Preincubate freshly isolated primary hepatocytes (2.10^7 cells)
for ~30 min at 37C in Krebs-HEPES-glucose buffer, as
described in Subheading3.1.1. - Spin down the cells in a 2-ml Eppendorf at 1200gfor 2 min
and permeabilize membrane by resuspending the cell pellet in
2 ml of KCl medium containing 100μg/ml digitonin. - Transfer the cells into the oxygraph chamber.
- Add 20μl of the respiratory-chain complex 1 substrates gluta-
mate/malate (stock: 0.5/0.25 M; final concentrations:
5/2.5 mM), or 20μl of the respiratory-chain complex 2 sub-
strate succinate/malate (stock: 0.5/0.25 M; final concentra-
tions: 5/2.5 mM), or 5.5μl of palmitoyl carnitine plus 8μlof
malate (stock palmitoyl carnitine/malate: 20 mM/0.25 M;
final palmitoyl carnitine/malate concentrations: 55 μM/
1 mM) with a Hamilton syringe through the cap of the oxy-
graph chamber (seeNote 11). - Start the acquisition of the basal (pseudo-state 4)JO 2 until
reaching a steady state, usually ~1–2 min (Fig.4).
Methods for Assessing Mitochondrial OXPHOS 281