AMPK Methods and Protocols

28. ECL Prime Western.


29. Re-blot plus strong antibody stripping solution (Millipore).

2.4 Preparation
of Islet RNA for RNA-
seq

1. Isolated islets.


2. 1.5 ml tubes.


3. Benchtop microcentrifuge (1.5 ml tubes).


4. TRIzol (Invitrogen).


5. Chloroform, molecular grade.


6. Isopropanol, molecular grade.


7. Ethanol, molecular grade.


8. Glycogen, molecular grade.


9. Sodium acetate.


10. Ultrapure, RNAse-free H 2 O.


11. DNase I, amplification grade (18068-015, ThermoFisher
    Scientific).

2.5 Impact of AMPK
on Glucose-Stimulated
Insulin Secretion
in Isolated Islets

1. Isolated islets (30 per condition).


2. KRBH stock solutions (can be kept for 1–2 months at 4C):
   l Mixed HEPES-bicarbonate (4): HEPES 40 mM, 8 mM
   NaHCO 3 , 40 mM NaCl.
   l Mixed salts (5): 650 mM NaCl, 18 mM KCl, 2.5 mM
   NaH 2 PO 4 , 2.5 mM MgSO 4 , 7.5 mM CaCl 2.


3. Krebs-Ringer Bicarbonate HEPES (KRBH) (1): This buffer
   is made fresh from the stock solutions above in water and
   equilibrated with 95% O 2 and 5% CO 2 for 10 min. pH is
   adjusted at 7.4 preceding supplementation with 0.1%
   (w/v) BSA.


4. KRBH-glucose: Prepare the solution of a range of glucose
   concentration (typically low glucose is 3 mM (KRBH-3 mM
   Glu) and high glucose is 17 mM or 11 mM (KRBH-17 mM
   Glu/KRBH-11 mM Glu), for mouse and human islets, respec-
   tively) and KCl (KRBH-30 mM KCl) in KRBH. KRBH-
   glucose solutions are kept at 37C.


5. 95% O 2 /5% CO 2 gas cylinders.


6. Nontreated 10 cm and 12-well plates.


7. Temperature and CO^2 -regulated cell incubator.


8. Water bath.


9. Zoom stereomicroscope, 10objective.


10. Benchtop microcentrifuge (1.5 ml tubes).


11. Lysis buffer: Acidic ethanol (75 ethanol: 23.5 H 2 O: 1.5 1 M
    HCl), 0.1% (v/v) Triton X-100.

418 Aida Martinez-Sanchez et al.