Calcium Imaging of Cortical Neurons using Fura-2 AM

Summary

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Abstract

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths. The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding. Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Protocol

Cell Culture

Cells can be grown using established techniques but must be plated on #1 glass coverslips coated with a cellular adhesive (like polylysine, polyornithine or laminin) to prevent the cells from detaching or moving during imaging experiments.

Solutions

Calcium imaging experiments can be performed using a variety of physiological solutions including cell culture media. It is important, however, to make sure that the solutions are free of phenol red, which greatly increases the fluorescent background. We use Tyrodes solution, which is easily made and mimics cerebrospinal fluid, an....

Discussion

In this presentation we've gone through all the steps for performing calcium imaging using Fura-2 AM. We used cortical neurons as a model but calcium imaging can be used to measure intracellular calcium in real time in a variety of cells. When doing this procedure it is important to use glass coverslips instead of plastic, since plastic is often fluorescent at ultraviolet wavelengths, which makes imaging difficult. Also, it is important to pre-coat the coverslips with polyornithine and laminin in order to prevent cells f.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
New Item	Fura-2 AM	Invitrogen	F1221	Protect from light
New Item	DMSO	Electron Microscopy Sciences	MX1457-6	Keep in dry place to prevent hydration
New Item	Albumin from bovine serum	Sigma-Aldrich	A8022-100G	Store at 4°
New Item	Imaging Chamber	Warner Instruments	Model RC-20H
New Item	Microscope	Nikon Instruments	Eclipse TE2000-U
New Item	Potassium chloride	Sigma-Aldrich	P9333-500G
New Item	Calibration Kit	Molecular Probes, Life Technologies	F6774	Protect from light