Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Alexander Hamilton; Elisa Vergari; Caroline Miranda; Andrei I. Tarasov

doi:10.3791/59491

JoVE Journal
Biology

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JoVE Journal Biology

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

마우스 췌도 세포의 하위 집단에 있는 화상 진찰 칼슘 역학

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08:03 min

November 26, 2019

DOI: 10.3791/59491-v

Alexander Hamilton^1,2, Elisa Vergari¹, Caroline Miranda³, Andrei I. Tarasov^1,4,5

¹Oxford Centre for Diabetes, Endocrinology and Metabolism,University of Oxford, ²Lund University Diabetes Centre, Unit of Molecular Metabolism, Clinical Research Centre,Malmö University HospitalMalmö, ³Institute of Neuroscience of Physiology, Department of Physiology, Metabolic Research Unit,University of Göteborg, ⁴Oxford National Institute for Health Research,Biomedical Research Centre, ⁵Life and Medical Sciences,University of Hertfordshire

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Overview

This study presents a method for imaging and quantifying calcium dynamics in pancreatic islet cells, allowing for the analysis of fluorescent signals in heterogeneous populations. The protocol enables real-time monitoring and yields high statistical power and reproducibility in the acquired data.

Key Study Components

Research Area

Calcium dynamics in cell populations
Pancreatic islet cell function
Quantitative imaging techniques

Background

The importance of pancreatic islet cells in glucose metabolism
Calcium signaling in cellular functions
Challenges of imaging heterogeneous cell populations

Methods Used

Imaging protocol using inverted microscopy
Fluorescent reporters for calcium dynamics
Immobilization of pancreatic islets for detailed imaging

Main Results

Observation of calcium spikes in alpha cells at low glucose levels
Responses of islet cell subpopulations to various stimuli like adrenaline and glutamate
Established methods for analyzing fluorescence intensity and cellular response

Conclusions

The study validates a reproducible imaging method for understanding calcium dynamics
This approach enhances understanding of pancreatic islet physiology and cellular interactions

Frequently Asked Questions

What is the significance of calcium dynamics in pancreatic islet cells?

Calcium dynamics are crucial for insulin secretion and overall metabolic regulation in pancreatic islet cells.

How does the imaging protocol improve data acquisition?

The protocol allows for real-time monitoring of cellular responses, enhancing statistical reliability and reproducibility.

What technologies are employed in the study?

Inverted microscopy and fluorescent reporters are essential for visualizing calcium dynamics in cells.

What are the applications of this research?

This protocol can be applied to studies of cellular signaling and interactions in various biological contexts, particularly diabetes research.

How are the fluorescent intensity data analyzed?

Data are normalized and analyzed for frequency of calcium spikes and responses to different chemical stimulants.

Can this method be adapted for other cell types?

Yes, the protocol can potentially be applied to other heterogeneous cell populations beyond pancreatic islets.

여기에서, 우리는 췌도 세포와 같은 이기종 세포 집단에 있는 칼슘 역학을 화상 진찰 그리고 정량화하기 위한 프로토콜을 제시합니다. 형광 기자는 그 때 고정화되고 심상되는, 그리고 형광 강렬의 역학의 세포 당 분석이 수행되는 작은 틀 내의 세포의 말초 층으로 전달됩니다.

이 프로토콜은 랑거한스의 췌장 섬 내의 작은 하위 집단의 급속한 격리된 기능을 허용합니다. 이 기술의 장점은 실시간 특성과 높은 통계적 능력, 따라서 획득 된 데이터의 높은 재현성을 포함한다. 이미징을 위한 섬을 고정하려면 반전된 현미경을 위해 이미징 챔버를 조립하고 챔버 내부에 유리 커버 슬립을 놓습니다.

유리 챔버 인터페이스가 방수되어 있는지 확인하고 커버 슬립이 현미경 목표의 범위 내에 있는지 확인하십시오. 다음으로, 20mm의 미세하고 거친 메쉬를 20mm 로 자르고 45 ~ 50 마이크로 미터의 두꺼운 끈적 끈적한 테이프를 사용하여 미세 한 메쉬 조각에 두 개의 스페이서 벽을 만듭니다. 거친 메쉬와 무게를 35mm 페트리 의 이미징 용액에 담그고 미세 메쉬를 해부 현미경 아래에 놓습니다.

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