AMPK Methods and Protocols

2 Materials and Reagents

Prepare all solutions using ultrapure water (mQ water; sensitivity

18 MΩ-cm at 25C) and analytical grade reagents. Prepare and

store all reagents at room temperature (unless indicated otherwise).

2.1 Materials
and Equipment

1. Primary mouse hepatocytes (seeNote 1).


2. M199 culture medium.


3. Fetal calf serum (FCS).


4. 30-ml Potter-Elvehjem.


5. Temperature-controlled centrifuge.


6. Ethanol absolute.


7. Dimethyl sulfoxide (DMSO).


8. Clark-type electrode device.


9. Hamilton syringes.


10. XFe96 extracellular flux analyzer (Seahorse Bioscience).


11. Seahorse 96-well plate and XF cartridge.

Fig. 1General approaches for monitoring mitochondrial respiration in cells or isolated organelles using Clark-
type oxygen electrode commercial devices. The oxygen consumption rates can be monitored in intact or
permeabilized cells (in situ mitochondria) and mitochondria isolated from tissues (e.g., liver) or cultured cell
lines (a) using various Clark-type oxygen electrode commercial devices (b) allowing continuous polarographic
measurement of oxygen concentration in sample medium

Methods for Assessing Mitochondrial OXPHOS 275