Islet Insulin Secretion Measurements in the Mouse

Alison Hugill1, Kenju Shimomura2, Roger D. Cox1

1 Mammalian Genetics Unit, Medical Research Council Harwell, Harwell Science and Innovation Campus, Oxfordshire, 2 Department of Medical Electrophysiology, Fukushima Medical University, School of Medicine, Fukushima
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/cpmo.14
Online Posting Date:  September, 2016
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Abstract

This article describes detailed protocols for in vitro measurements of insulin function and secretion in isolated mouse islets for the analysis of glucose homeostasis. We specify a method of enzyme digestion and hand picking to isolate and release the greatest number of high quality islets from the pancreas of the mouse. We describe an effective method for generating dynamic measurements of insulin secretion using a perifusion assay including a detailed protocol for constructing a peristaltic pump and tubing assembly. In addition we describe an alternative and simple technique for measuring insulin secretion using static incubation of isolated islets. © 2016 by John Wiley & Sons, Inc.

Keywords: islets of Langerhans; insulin secretion; perifusion; dynamic measurements; static measurements

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Islet Isolation
  • Basic Protocol 2: Dynamic Measurements Using Perifusion Assay
  • Support Protocol 1: Peristaltic Pump and Tubing Assembly
  • Basic Protocol 3: Static Measurements of Islet Insulin Secretion
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Islet Isolation

  Materials
  • Dissociation buffer (see recipe)
  • Quenching buffer (see recipe)
  • Culture medium (see recipe)
  • Ice
  • Mouse (strain C57BL/6NTac was used in this example)
  • 70% (v/v) ethanol
  • Dissecting board and pins
  • Dissecting scissors
  • Serrated dissecting tweezers (thumb forceps: two pair)
  • Curved mosquito forceps
  • Straight mosquito forceps
  • 2‐ml syringe
  • 30.5‐gauge needle
  • Dissecting needle
  • Stereo microscope
  • Light source
  • Spring scissors
  • 15‐ml tube
  • Timer
  • 35‐mm culture dishes
  • P20 pipet and tips
  • 37°C incubator with humidified 5% CO 2 in air
NOTE: Buffers should be kept on ice and culture media equilibrated to 37°C. Inflating the pancreas takes some practice, so it is worth practicing the technique on spare mice using water instead of dissociation buffer, until competent.

Basic Protocol 2: Dynamic Measurements Using Perifusion Assay

  Materials
  • 5% (v/v) bleach
  • Sterile distilled H 2O (sdH 2O)
  • 2 mM glucose solution (see recipe)
  • 20 mM glucose solution (see recipe)
  • Tolbutamide solution (see recipe)
  • Acid/ethanol (see recipe)
  • 50 equal‐sized, isolated islets (cultured overnight; from protocol 1)
  • Mouse insulin ELISA
  • Tweezers
  • 13‐mm, 8.0‐μm membrane filters (2, 1 filter/holder)
  • P200 pipet and tips
  • Stereo microscope
  • Light source
  • Stopwatch
  • Clamp stand
  • 1.5‐ml tubes (labeled 1 to 40 for each mouse)
  • 1.5‐ml tube racks
  • 2‐ml tubes
NOTE: Glucose and tolbutamide solutions should be equilibrated to 37°C before experimentation. Set up peristaltic pump and tubing assembly as described in protocol 3Support Protocol. In this protocol, the assembly set up is for two mice. Each experiment must contain a control animal. Pooling data from at least three biological replicates is recommended for robust results.

Support Protocol 1: Peristaltic Pump and Tubing Assembly

  Materials
  • Sterile distilled H 2O (sdH 2O)
  • Flexible tubing, ID 1.6 mm, OD 3.2 mm (≥400 cm)
  • Male luer fittings for 1.6‐mm ID tubing (13 total)
  • Three‐port manifold
  • Three‐port infusion Y swivel thread
  • Female luer fittings for 1.6‐mm ID tubing (6 total)
  • Peristaltic pump (with ≥2 channels and variable speed allowing a flow rate of 1 ml/min)
  • Slide clamps for 3.2‐mm OD tubing (4 total)
  • 13‐mm filter holder (islet chamber) with inlet and outlet connections (2, 1/mouse)
  • Silicone gasket for 13‐mm filter holder (2, 1 gasket/holder)
  • Timer

Basic Protocol 3: Static Measurements of Islet Insulin Secretion

  Materials
  • 2 mM glucose solution (see recipe)
  • 20 mM glucose solution (see recipe)
  • Tolbutamide solution (see recipe)
  • Acid/ethanol (see recipe)
  • Equal‐sized, isolated islets (cultured overnight; from protocol 1)
  • Mouse insulin ELISA
  • P20 and P200 pipet and tips
  • 24‐well tissue culture plates
  • Stereo microscope
  • Light source
  • 37°C incubator with humidified 5% CO 2 in air
  • Stopwatch
  • 96‐well plate
NOTE: Glucose and tolbutamide solutions should be equilibrated to 37°C before experimentation. Each experiment must contain a control animal. Pooling data from at least four biological replicates is recommended for robust results.
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Figures

Videos

Literature Cited

Literature Cited
  Animals (Scientific Procedures) Act. 1986. c. 14. (http://www.legislation.gov.uk/ukpga/1986/14). Accessed 2016.
  Ferrannini, E. and Mari, A. 2014. β‐Cell function in type 2 diabetes. Metab. Clin. Exp. 63:1217‐1227. doi: 10.1016/j.metabol.2014.05.012.
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  Shimomura, K., Galvanovskis, J., Goldsworthy, M., Hugill, A., Kaizak, S., Lee, A., Meadows, N., Quwailid, M.M., Rydstrom, J., Teboul, L., Ashcroft, F., and Cox, R.D. 2009. Insulin secretion from β‐cells is affected by deletion of nicotinamide nucleotide transhydrogenase. Meth. Enzymol. 457:451‐480. doi: 10.1016/S0076‐6879(09)05025‐3.
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