AMPK Methods and Protocols

• Buffer A: 0.8% (v/v) HCl.
  
  
  • Buffer B: 0.1 M NaOH.
  
  
  • Buffer C: 50 mM NaH 2 PO 4 , 25 mM NaCl, adding
    Na 2 HPO 4 to give pH 5.2.
  
  
  • Buffer D: 0.1 M MES monohydrate/Tris pH 5.2, 0.2%
    (w/v).
    Hydroxyethyl cellulose, 0.2% (w/v), is added to buffers C and
    D(seeNote 6).
    We use a capillary of length 31.2 cm, diameter 75μm, and
    aperture size 100
    800 μm.
  
  
  
  

10. LC-MS instrument. We use a TSQ Quantiva interfaced with an
    Ultimate 3000 Liquid Chromatography system, equipped with
    a porous graphitic carbon column and using the following
    buffers:

• Buffer A: 0.3% (v/v) formic acid adjusted to pH 9 with
  ammonia prior to a 1:10 dilution.


• Buffer B: 80% (v/v) acetonitrile.

2.7 Analysis
of Cellular Oxygen
Uptake Using
an Extracellular Flux
Analyzer

1. Seahorse Extracellular Flux Analyzer (e.g., XF24).


2. Seahorse XF Base Medium.
   3.D-Glucose.


3. Sodium pyruvate.
   5.L-Glutamine.


4. Seahorse XF Sensor Cartridge.


5. Seahorse XF Cell Culture plate.


6. Seahorse XF Calibrant solution.


7. Phenformin (or similar compound as a positive control).


8. Uncoupler (e.g., 2,4-dinitrophenol).


9. Compound of interest.

3 Methods

3.1 Methods
of Plating and Treating
Adherent Cells

1. Plate cells in 6 cm dishes in 4 ml of appropriate media supple-
   mented with 10% FBS. Depending on the cell type, media may
   be changed for one with a more physiological glucose concen-
   tration (5 mM) 16 h prior to treatment, when cells are70%
   confluent (seeNote 7).


2. Activating compounds are made up as described by suppliers.
   Usually compounds are dissolved in dimethyl sulfoxide
   (DMSO) and are then added such that the final concentration
   of DMSO in the culture medium is no greater than 1%. Higher
   concentrations of DMSO, or incubation periods longer than

244 Simon A. Hawley et al.