- Incubate the pancreas in a water bath at 37C for exactly
10 min (seeNote 6). - Add 15 ml of RPMI and shake vigorously for ~20 s. The
pancreas should be then fully dissociated with very few clumps.
Spin down at 200g for 1 min. - Discard supernatant and wash with 15 ml of RPMI. Spin down
at 200gfor 1 min. Repeat this step an additional two times. - Resuspend the pellet in 4 ml of Histopaque 1119, mixing by
hand. Transfer the resulting homogenous milky solution into a
15 ml tube. - Drop by drop, add 4 ml of Histopaque 1083 to create a density
gradient. - On top, add 4 ml of RPMI, also drop by drop.
- Spin at 1200gin a centrifuge set with maximum accelera-
tion and minimum deceleration for 20 min, at room tempera-
ture (RT). - Once spun, the islets are found in a ring between the RPMI and
Histopaque 1083 layers. Remove the islets using a plastic
Pasteur pipette and place into a 10 cm nontreated plate with
8 ml of mouse islet medium. At this point we recommend the
use of a 100μm nylon mesh cell strainer to separate the islets
by size. - To eliminate all the remaining Histopaque (seeNote 7), hand-
pick the islets into a new plate containing mouse islet medium.
Maintenance of Pancreatic Islets- The islets are maintained in a 5% CO 2 incubator at 37Cin
mouse islet medium at least overnight before performing any
downstream experiments (seeNotes 8– 10 ).
3.2 Approaches
to AMPK Modulation
in Islets
Besides genetically and by manipulation of glucose concentration in
the medium islet, AMPK activity can be modulated pharmacologi-
cally or through viral transduction (seeNote 11).
Pharmacological Modulation of AMPK Activity
Pharmacological AMPK activators can be used to assess the
effect of “short-term” (i.e., 1.5–2 h), which normally correspond
to the duration of an in vitro GSIS experiment (seeSubheading3.3)
or “long-term” (24–48 h) AMPK activation in islet function, as
detailed below:- Prepare 6-well plates (for suspension culture, to avoid the
attachment of the islets to the surface) with 2.5–3 ml of
mouse islet medium containing 20μM C-13, 50μM C-991,
or both (concentrations chosen to give close to maximal effects
on AMPK activity, unpublished data and [22]). A control well
is prepared containing an equivalent amount of diluent
(DMSO).
420 Aida Martinez-Sanchez et al.