- Add to islet pellet 1 ml of lysis buffer.
- Sonicate the islets in lysis buffer for 10 s at 10 W, then place
on ice. - Proceed to insulin level measurement or store at 20 Cor
lower. - Insulin levels analysis by HTRF Cisbio Insulin Ultra-Sensitive
Kit. The principle is based on homogeneous time-resolved
fluorescence (HTRF) technology in which the insulin is
detected in a sandwich assay format using two different mono-
clonal antibodies (one donor and one acceptor) [34]. Dilute
sample with assay buffer (KRHB without glucose), no dilution
for low glucose condition of secreted samples, 1:3 for high
glucose and KCl conditions of secreted insulin samples, and
1:200 for samples containing total islets in order to fit in the
standard range of HTRF kit (0.03–8 ng/ml).
4 Notes
- Clamp as far inside the intestine as desired, but avoid obstruct-
ing the duct outside the intestine which would result in the
blockage of the collagenase flow into the pancreatic area. - Inserting the needle in the junction will aid needle penetration
into the duct and will allow for space to reintroduce the needle
if failure. Introduce ~0.5 cm of the needle inside the duct and
keep constant pressure. If the needle has been adequately
located inside the duct, resistance should be encountered
when injecting. If a very localized site of inflation is observed,
chances are that the needle has been inserted outside the duct,
probably between the duct and the fat that often surround the
duct. Patiently removing that fat with the aid of the needle
before attempting the insertion inside the duct might help the
less experienced researcher to succeed. - The degree of inflation will largely correlate with the yield of
isolated islets. It is important for the collagenase to reach the
whole pancreatic surface. - In bigger animals, such as CD1 mice, it might be useful to
clamp the duct entry to the liver, to minimize the amount of
collagenase leaking inside this organ. This is not necessary if the
needle used is thick enough to prevent the reflow of collagenase
in the liver direction. - It is safe to leave the pancreas on ice for 2–3 h if necessary.
- The length of the incubation might vary with the collagenase
used and should be optimize for each type of collagenase/
batch.
426 Aida Martinez-Sanchez et al.