AMPK Methods and Protocols

5. Transfer the tube to the EPR machine and start the measure-
   ment. Figure1 shows representative EPR tracings from murine
   aortic rings treated with and without angiotensin II, a potent
   stimulator of ROS production that results in diminished vascu-
   larlNO levels.

3.2 Amplex Red
Assay for the Detection
of Hydrogen Peroxide

This is an HPLC method. You should prepare all solutions using

ultrapure water and analytical grade reagents.

1. Remove all connective tissue as well as vascular fat from aortic
   sections. Cut the aorta into pieces of 3 mm in length. One
   sample consists of two pieces (seeNote 7).


2. Transfer two pieces of the aortic sections to one 1.5 mL tube;
   add 150μL of master mix. Make sure that the sections are
   completely covered with solution.


3. Incubate the samples for 20 min on a shaker at 37C, 500 rpm.

Copyright © 2014, Mary Ann Liebert, Inc.

3249 3306 3363

Magnetic Field [G]

Intensity

WT Ctr

+ATII

Fig. 1NO quantification by EPR using Fe(II)(DETC) 2. Representative EPR spectra

of aortic sections; angiotensin II in vivo treatment was used to establish

endothelial dysfunction with diminished NO bioavailability. EPR-based NO anal-

ysis from untreated mouse aorta shows the characteristic triplet signal (high-

lighted by the red arrows), which is significantly weaker after in vivo angiotensin

II treatment (highlighted by the blue arrows). Figure modified from [8], Copyright

©2014, Mary Ann Liebert, Inc.

500 Swenja Kro ̈ller-Scho ̈n et al.