AMPK Methods and Protocols

analytical grade reagents. Prepare and store all reagents at room

temperature (unless indicated otherwise). Diligently follow all

waste disposal regulations when disposing waste materials.

2.1 Vascular Nitric
Oxide Analysis by EPR
Spectroscopy with Fe
(II)(DETC) 2
as Spin Trap

1. Argon gas.


2. Phosphate buffered saline (PBS): 145 mM NaCl, 2.7 mM KCl,
   1.5 mM KH 2 PO 4 , 8 mM Na 2 HPO 4 , pH 7. Aerate PBS in a
   conical tube for 30 min with argon gas.


3. FeSO 4 solution: 1.49 M FeSO 4. Weigh 3.4 mg of Fe(II)SO 4
   and put it in a 50 mL conical tube. Add 15 mL of the PBS
   solution to the FeSO 4 and mix thoroughly. The FeSO 4 has to
   be dissolved completely. Aerate the solution for another 30 min
   with argon gas.


4. DETC solution: 1.62 M DETC. Weigh 5.4 mg of DETC
   (diethyldithiocarbamic acid) and put it in a 50 mL conical
   tube. Add 15 mL of PBS to the DETC and mix thoroughly.
   The DETC has to be resolved completely. Aerate the solution
   for another 30 min with argon gas.


5. Krebs-Hepes buffer (KH): 99.01 mM NaCl, 4.69 mM KCl,
   2.5 mM CaCl 2 , 1.2 mM MgSO 4 , 25 mM NaHCO 3 , 1.03 mM
   K 2 HPO 4 , 20 mM sodium-Hepes, 11.1 mMD-glucose, adjust
   pH to 7.35 (seeNote 1).


6. Ca ionophore: 10 mM Ca ionophore. Ca ionophore stock
   solution solved in dimethyl sulfoxide (DMSO) should be
   thawed and stored at room temperature till usage.


7. 1 mL syringes: remove the small end of 1 mL syringes and the
   small end of the plunger inside of the syringe. Label every
   syringe according to the treatment group.


8. EPR machine (e.g., Magnettech, Berlin).


9. Plastic ware: 1 m syringes (removed caps).

2.2 Amplex Red
Assay for the Detection
of Hydrogen Peroxide

1. Master mix: 20μM myxothiazol, 100μM Amplex red, 0.2μM
   horseradish peroxidase (HRP). For preparation to 250μLof
   PBS (w/o Ca/Mg), add 1.25μL of myxothiazol (4 mM in
   DMSO), 0.5μL of Amplex red solution (100μM), and 0.5μL
   of HRP (100μM). Prepare master mix in a conical tube
   (volumes are meant per sample!).


2. H 2 O 2 standard solution: 10μMH 2 O 2 (30% H 2 O 2 ¼10 M) in
   150 μL master mix.


3. Buffer sample: 250μL of PBS in a HPLC vial.


4. Organic solvent: 90% acetonitrile/10% water (v/v).


5. Mobile phase: 50 mM citrate buffer, pH 2.2.


6. HPLC setup: we are using a system that consists of a control
   unit, two pumps, a mixer, detectors, a column oven, a degasser,

NO & Oxidative Stress 497