Name: calcium imaging of cortical neurons using fura-2 am
Uploaded: 2009-01-19T00:00:00
Description: Watch this Scientific Journal Video about Calcium Imaging of Cortical Neurons using Fura-2 AM at JoVE.com

<p class="jove_title">Cell Culture</p>
<p class="jove_content">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.</p>
<p class="jove_title">Solutions</p>
<p class="jove_content">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 fr...

<ol>
	<li>Grynkiewicz, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+generation+of+Ca2++indicators+with+greatly+improved+fluorescence+properties.">A new generation of Ca2+ indicators with greatly improved fluorescence properties.</a> <em style='font-style: italic'>J. Biol. Chem</em>. <b>260</b>, 3440-3450 (1985).</li><li>Lewis, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen-induced+oscillations+of+cytosolic+Ca2++and+transmembrane+Ca2++current+in+human+leukemic+T+cells.">Mitogen-induced oscillations of cytosolic Ca2+ and transmembrane Ca2+ current in human leukemic T cells.</a> <em style='font-style: italic'>Cell Regul</em>. <b>1</b>, 99-112 (1989).</li><li>Dolmetsch, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+between+intracellular+Ca2++stores+and+depletion-activated+Ca2++channels+generates+[Ca2+]i+oscillations+in+T+lymphocytes.">Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes.</a> <em style='font-style: italic'>J Gen Physiol</em>. <b>103</b>, 365-388 (1994).</li></ol>

<p class="jove_content">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...

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<td>New Item</td>
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<td>Sigma</td>
<td>A8022-100G</td>
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<td>New Item</td>
<td>Imaging Chamber</td>
<td>Warner Instruments</td>
<td>Model RC-20H</td>
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<td>Nikon</td>
<td>Eclipse TE2000-U</td>
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calcium imaging of cortical neurons using fura-2 am

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.

Watch this Scientific Journal Video about Calcium Imaging of Cortical Neurons using Fura-2 AM at JoVE.com

Calcium Imaging of Cortical Neurons using Fura-2 AM

<p class="jove_content">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.</p>

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

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