Lepr<sup>db</sup> Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Oanh H. Do; Jiun T. Low; Peter Thorn

doi:10.3791/52632

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Phot...

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Lepr^db Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

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10:09 min

May 11, 2015

DOI: 10.3791/52632-v

Oanh H. Do¹, Jiun T. Low¹, Peter Thorn¹

¹School of Biomedical Sciences,The University of Queensland

Overview

This protocol details the isolation of islets from the Lepr db mouse model of type 2 diabetes and a live-cell assay for measuring insulin secretion using two-photon microscopy.

Key Study Components

Area of Science

Neuroscience
Endocrinology
Cell Biology

Background

Type 2 diabetes is characterized by impaired insulin secretion.
Islets of Langerhans are critical for insulin production.
Two-photon microscopy allows for detailed imaging of cellular processes.
Understanding insulin secretion mechanisms is vital for diabetes research.

Purpose of Study

To isolate viable islets from diabetic mice.
To assess insulin secretion from beta cells within the islets.
To utilize advanced imaging techniques for real-time analysis.

Methods Used

Preparation of enzyme mixture for pancreas digestion.
Injection of enzyme into the pancreas of euthanized mice.
Separation and culturing of islets from pancreatic digest.
Application of two-photon microscopy to image insulin granule fusions.

Main Results

Successful isolation of living islets from Lepr db mice.
Effective imaging of insulin secretion events in real-time.
Insights into the regulation of insulin release from beta cells.
Potential implications for understanding diabetes pathology.

Conclusions

This protocol enhances the understanding of insulin secretion mechanisms.
It provides a valuable tool for diabetes research.
Future studies can build on these findings to explore therapeutic strategies.

Frequently Asked Questions

What is the significance of isolating islets from diabetic mice?

Isolating islets allows researchers to study insulin secretion mechanisms in a controlled environment, which is crucial for understanding diabetes.

How does two-photon microscopy contribute to this research?

Two-photon microscopy provides high-resolution imaging of live cells, enabling real-time observation of insulin granule fusions.

What are the main challenges in isolating islets?

Challenges include ensuring the viability of islets and minimizing damage during the isolation process.

Can this protocol be applied to other models of diabetes?

Yes, with modifications, this protocol can be adapted for other diabetic models.

What future research directions could stem from this study?

Future research could explore therapeutic interventions targeting insulin secretion pathways in diabetes.

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Lepr^dband details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

The overall goal of this procedure is to obtain viable living eyelets from the diabetic LEP R DB mice, and assess insulin secretion from beta cells within the eyelets using live cell to photon imaging. This is accomplished by first preparing reagents such as the enzyme mixture for the pancreas digestion, the culture media for eyelet culturing, and the extracellular buffer for the two photon assay. Next, the abdomen of a euthanized mouse is opened up and the enzyme mixture is injected into the pancreas to digest the organ.

Then the eyelets are separated from the pancreatic digest and cultured. Finally, two photon microscopy is used to image single insulin granule fusions from beta cells within the eyelet. Ultimately, this protocol will help researchers to understand how insulin secretion is controlled in health and disease.

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