would be an important confounder in evaluating the effects of
AMPK activation on cellular function, as it would act as an
AMPK activator even in the control incubation conditions.
- This solution is prepared from the four stock solutions (A–D):
Add 25 mL of each stock solution (A–D) to a 1 L glass beaker,
adding water to final volume of ~400 mL and then adding
0.9 g ofD-glucose to the final solution. Bring the solution to
37 C by placing it on a water bath. Then equilibrate the
solution by bubbling through it 5% CO 2 (balance air) while
keeping it at 37C for ~15 min. Then check the pH of this
solution. It should be close to 7.4 without major adjustments.
- This Hank’s Balanced Salt Solution must be prepared using a
volumetric flask adding water first and carefully adding the
different reagents from stock solutions and adding the calcium
containing stock solution last to prevent calcium phosphate
precipitation [12, 18].
- Either a peristaltic or a syringe pump will work. Speed of
perfusion pump is ~4.0 mL/min. One end of the tubing
should be connected to a needle to be inserted into the left
ventricle and the other end of the tubing to be submerged into
a container of warm (37C) CO 2 -equilibrated Ringer buffer.
Alternatively, the perfusion pressure could be equilibrated with
the animal’s blood pressure to prevent changes in the kidney
due to perfusion pressures that are either too low or too high.
- There should be as many tubing pieces for the gas mix as there
are condition vials, plus an additional four tubing pieces to
allow equilibration of slice holding containers.
- We have not used metformin in this system as it requires hours
to days to induce AMPK activation.
- Prepare the fixative in PBS fresh every time on the day of the
slice preparation. Do not reuse the fixative, and do not save for
another slice preparation at a later date. Please discard accord-
ing to laboratory safety department policies.
- Lysis solution detailed preparation: 100μL of 1 M Tris–HCl,
pH 7.4; 1.6 mL of 1 M NaCl solution; 5μL of IGEPAL
CA-630 (Sigma-Aldrich #1-3021); 100μL of Triton X-100
(Sigma-Aldrich #T8787); 20μL of 0.5 M EDTA; 50μLof
0.2 M EGTA; one complete protease inhibitor tablet—Roche
complete mini, EDTA-free (Sigma-Aldrich #11836170001);
one PhosStop tablet (Roche); 50μL of 100 mg/mL AEBSF
(Pefabloc, Sigma-Aldrich #76307); add pure water up to
10 mL; use a stir bar to dissolve ingredients at RT.
- Until the method is established in the laboratory, the presence
of two people throughout the procedure decreases the chances
of mishaps.
AMPK Studies using Kidney Slices 461
