2 Materials
2.1 Isolation
and Maintenance
of Pancreatic Islets
- Dissection tools: Surgical scissors (blunt/blunt and sharp/
blunt), Adson forceps (12 cm), and pean hemostatic clamp
artery forceps (straight or curved). - 70% (v/v) ethanol.
- 5 and 50 ml syringes.
- Nontreated 10 cm plates.
- 30 G needles.
- 15 and 50 ml conic tubes.
- 100μm nylon mesh cell strainer.
- Plastic Pasteur pipettes.
- Water bath.
- Benchtop centrifuge (for 15 and 50 ml tubes).
- Zoom stereomicroscope, 10objective.
- Temperature and CO 2 -regulated cell incubator.
- Histopaque 1119 and 1083.
- Collagenase solution: 1 mg/ml collagenase (Ref 17,456,
SERVA) prepared in RPMI. Sterilize by filtration with a
0.22μm pore size filter. - Mouse islet medium: RPMI (containing 11 mM glucose), 10%
(v/v) fetal bovine serum (FBS), 1% (v/v) penicillin/
streptomycin.
2.2 Reagents
for Modulation
of AMPK Activity
- Isolated islets.
- Nontreated 6-well plates.
- 1.5 ml tubes.
- Mouse islet medium (seeSubheading2.1,item 10).
- Dimethyl sulfoxide (DMSO), molecular grade.
- AMPK pharmacological activators.
- Benchtop microcentrifuge (1.5 ml tubes).
2.3 Measurement
of AMPK Activity
in Isolated Islets
SAMS Peptide Assay- Isolated islets.
- Ice-cold PBS with the appropriate glucose concentration.
- Ice-cold lysis buffer: 50 mM Tris–HCl (pH 7.4 at 4 C),
250 mM sucrose, 50 mM NaF, 1 mM Na pyrophosphate,
1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM benzami-
dine, 0.1 mM PMSF, 5μg/ml soybean trypsin inhibitor, and
1% (v/v) Triton X-100.
416 Aida Martinez-Sanchez et al.