Single-Cell Calcium Imaging for Studying the Activation of Calcium Ion Channels

Summary

This article presents a method for real-time, quantitative monitoring of calcium ion (Ca²⁺) concentrations in cells using single-cell Ca²⁺ imaging with the Fura-2/AM dye. This technique enables efficient dye loading and accurate calculation of Ca²⁺ levels through fluorescence intensity ratios, making it a simple and rapid approach for research applications.

Abstract

Single cell Ca²⁺ imaging is essential for the study of Ca²⁺ channels activated by various stimulations like temperature, voltage, native compound and chemicals et al. It primarily relies on microscopy imaging technology and the related Ca²⁺ indicator Fura-2/AM (AM is the abbreviation for Acetoxymethyl ester). Inside the cells, Fura-2/AM is hydrolyzed by esterases into Fura-2, which can reversibly bind with free cytoplasmic Ca²⁺. The maximum excitation wavelength shifts from 380nm to 340nm (when saturated with Ca²⁺) upon binding. The emitted fluorescence intensity is quantitatively related to the concentration of bound Ca²⁺. By measuring the 340/380 ratio, the Ca²⁺ concentration in the cytoplasm can be determined, eliminating errors caused by variations in the loading efficiency of the fluorescent probe among different samples. This technology allows for real-time, quantitative, and simultaneous monitoring of Ca²⁺ changes in multiple cells. The results are stored in “.XLSX” format for subsequent analysis, which is fast and generates intuitive change curves, greatly improving the detection efficiency. From different experimental perspectives, this article lists the use of this technology to detect Ca²⁺ signals in cells with endogenous or overexpressed channel proteins. Meantime, different methods for activating cells were also showed and compared. The aim is to provide readers with a clearer understanding of the usage and applications of single cell Ca²⁺ imaging.

Introduction

Ca²⁺ plays a crucial role in cellular signal transduction, regulating various cellular functions such as muscle contraction¹, nerve conduction², secretion³, and gene expression⁴, thereby influencing multiple physiological processes. Abnormal Ca²⁺ concentrations can lead to diseases such as arrhythmias⁵, coagulation disorders⁶, and hormonal imbalances⁷. Therefore, studying the mechanisms of intracellular Ca²⁺ concentration changes is of paramount importance.

Protocol

The experimental methods were approved by and followed the IACUC guidelines of Tsinghua University and Beijing University of Chinese Medicine. This protocol introduces single-cell Ca²⁺ imaging methods for various cell types, including primary keratinocytes isolated from the skin of several newborn mice (within three days of birth, with sex-randomized littermates, C57BL/6 mice). Details of the reagents and equipment used in this study are listed in the Table of Materials.

Discussion

The application of single-cell Ca²⁺ imaging systems is extensive, enabling the study of Ca²⁺ signals in various cell types, including keratinocytes, stem cells¹⁶, liver cells, heart cells¹⁷, podocytes¹⁸, immune cells, and cell lines overexpressing target proteins^10,19. This technique measures changes and absolute values of cellular Ca²⁺ concentrations and play.......

Materials

Name	Company	Catalog Number	Comments
Camera	Nikon
Capsaicine	Sigma	211275
CL-100 temperature controller	Warner Instruments
Cyclopiazonic Acid (CPA)	Sigma	C1530
DG-4 light	Sutter Instrument Company
Dimethyl sulfoxide (DMSO)	Amresco	231
DPBS	Thermofisher	14190144
Fluorescence imaging software (MetaFluor, Paid software)	Molecular Devices
Fluorescence microscope	Nikon
Fura-2/AM	Invitrogen	F1201
HBSS buffer	Gibco	14175103
HEPES	Sigma	H3375
Lipofectamine 3000	Invitrogen	L3000008
Pluronic F-127	Beyotime	ST501
poly-D-lysine	Beyotime	ST508
SC-20 liquid circulation heating/cooling device	Harvard Apparatus
White-light source	Nikon