- The islets can be first placed in a fresh tube and washed by
adding islet media into the tube, spinning during 2 min at
1000 rpm, and afterward resuspended in fresh mouse islet
media and placed in a 10 cm nontreated plate to skipsteps
15 and 16. Nevertheless, we have found that, due to Histopa-
que being carried with the islets, the islets sometimes don’t
pellet adequately at the bottom of the 15 ml tube, in which case
some of the islets are lost. - This is to allow recovery from the collagenase digestion. Also,
note that the mouse islet medium contains 11 mM glucose,
which corresponds to the concentration found in plasma in
fed mice. - Isolation, manipulation, and maintenance of islets in vitro
might lead to undesired AMPK activation. First, any contami-
nation of fresh rodent islet preparations with exocrine tissue
will markedly increase the measured AMPK activity. This is
consistent with the higher levels of the enzyme in the exocrine
tissue [24]. Secondly, very large islets have a hypoxic central
core [35] and doubtless high AMPK activity. Maintenance of
islets in culture for3 days before the assay was found to
overcome this problem, probably by allowing time for contam-
inating exocrine tissue and the hypoxic central cores of large
islets, to necrose [36]. Another way of avoiding the problem is
to either handpick the islets or filter the islets preparation on a
100 μM cell strainer and only use the small ones for the assay.
This is particularly important if overexpressing adenoviruses as
they will not infect the core of large islets [37]. Nevertheless,
this might not be possible when a high amount of islet material
is required for downstream applications (such as measurement
of activity with SAMS peptide assay). And thirdly, washing the
islets in PBS before protein isolation can activate AMPK, this
can be prevented by adding glucose in the PBS at the same
concentration as in the culture conditions or by strictly using
ice-cold PBS and keeping the samples on ice at all times. - Human pancreatic islets are generally provided to our lab from
several research groups around the world. Human islets are
maintained in medium containing a low glucose concentration
(5.5 mM glucose), which mimics blood glucose concentration
after a meal. Human islet medium: RPMI without glucose (add
glucose up to 5.5 mM), 10% (v/v) FBS, 1% (v/v) penicillin/
streptomycin, and 2.5 mg/L amphotericin B. - The potent and selective AMPK activators compound 13 and
compound 991 were described previously [16, 17]. AMPK-
DN and AMPK-CA virus [12] are available from our lab. - The number of islets to be used will greatly vary depending on
the sensitivity of the downstream application. We have used as
Manipulation and Measurement in Islets 427