Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Summary

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Abstract

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from β-cells), glucagon (α-cells) and somatostatin (δ-cells). β-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, α-cells and δ-cells (making up around 20% (30%) and 4% (10%) of the total rodent¹ (human²) islet cell numbers, respectively) is less predictable and is therefore of special interest.

Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of β-cells is minimized.

Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.

Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Introduction

The purpose of the method is to image real-time changes in cytosolic calcium concentration ([Ca²⁺]_cyt) in minor subpopulations of pancreatic islet cells. This allows uncovering the mechanisms governing hormone secretion in these cells, revealing details about the cross-talk between different cell types and, potentially, introducing a populational dimension into the larger picture of islet signaling.

Islets consist of several cell types. Besides the more well-known insulin-secreting β-cells, there are at least two subpopulations that are also critical in regulating blood glucose³. α-Cel....

Protocol

All methods described here were developed in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986) and the University of Oxford ethical guidelines.

1. Isolate mouse pancreatic islets

1. Prepare the culture medium and the isolation solution.
   1. Make up the culture medium: RMPI1640 (see Table of Materials), supplemented with 10% of fetal calf serum, 100 units/mL penicillin, 100 μg/mL streptomycin. Dispense the medium into two 60 mm plastic Petri dishes (not treated with any adhesive), and keep the dishes in the incubator (37 °C, 5% CO₂, absolute humidity).
   ....

Results

Islets load fairly well with the trappable dyes (Figure 1A), unless the lipid composition of the membrane has been affected (e.g., by chronic exposure to fatty acids). The human adenovirus type 5 (Ad5) vector also targets all islet cells (Figure 1B). Problems may arise when more than one recombinant sensor is being expressed in the same cell. Furthermore, islets are typically very well immobilized using the technology described .......

Discussion

There are three stages in the protocol that are critical for overall success. Successful injection of the Liberase enzyme into the bile duct determines not just the quantitative success of the isolation procedure but also impacts the quality of the isolated islets. Uninflated pancreata may result in lack of some important metabolic responses in the isolated islets. Secondly, the loading of the dye/the expression of the sensor defines the signal-to-noise ratio of the time-lapse recording. Signals are absent or attenuated .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
40x/1.3 objective
Axiovert 200 microscope
emission
Excitation
Fetal bovine serum	Sigma-Aldrich	F7524-500ML
Fluo4	Thermo Fisher (Life Technologies)	F14201
GCaMP6f, in (human type 5) adenoviral vector	Vector Biolabs	1910
Hanks' solution	Thermo Fisher (GibCo, Life Technologies)
Liberase	Sigma-Aldrich	5401020001
penicillin/streptomycin	Thermo Fisher (GibCo, Life Technologies)	15140122
RPMI medium	Thermo Fisher (GibCo, Life Technologies)	61870044
Zeiss LSM510-META confocal system	Carl Zeiss