AMPK Methods and Protocols

4. After 20 min remove the samples from the heater at the same
   time, and transfer the solution to the HPLC vials. Make sure
   that the aortic sections remain in the 1.5 mL tube.


5. Measure the standard, the buffer sample, and your unknown
   samples in the HPLC machine using the following settings: a
   high-pressure gradient is employed with the organic solvent
   B(90 vv% acetonitrile/10 vv% water) and aqueous solvent
   A(50 mM citrate buffer pH 2.2) as mobile phases with the
   following percentages of the organic solventB: 0 min, 41%;
   7 min, 45%; 8–9 min, 100%; 10–12 min, 41% (linear gradient
   increases within the indicated time windows). The remaining
   percentages of the mobile phase are the aqueous bufferB. The
   flow should be 1 mL/min, compounds are detected by their
   absorption at 300 nm, and resorufin is also detected by fluores-
   cence (Ex. 570 nm/Em. 590 nm). Inject 50μL of the sample
   to the HPLC system. Typical retention time of resorufin is
   2.8 min, and its formation is sensitive to the presence of catalase
   (Fig.2).

3.3 Dihydroethidium
(DHE) Staining
for the Detection
of Superoxide Anions
in Vascular
Cryosections

DHE is a UV-sensitive compound. For the next method, it is

necessary to use dark reaction cups, and every step using the

compound or the solutions should be done in a darkened room.

0

10

20

30

40

50

Response [mV]

0 1 2 3 4 5

Time [min]

100μM amplex red

+0.1μM HRP in HX buffer

+48 mU/ml XO

+48 mU/ml XO

+ PEG-Cat (800 U/ml)

oxidized

amplex red

Fig. 2Amplex red/HRP assay: HPLC tracings. Characteristic HPLC tracings

obtained from the Amplex red/HRP assay. The red line shows the negative

control (only hypoxanthine added); the blue line shows the oxidized Amplex

red signal (ROS generated in vitro by addition of xanthine oxidase) at the

retention time of 2.8 min. Addition of PEG catalase in order to remove H 2 O 2

from the system diminished the signal (green line)

NO & Oxidative Stress 501