Name: imaging local ca2+ signals in cultured mammalian cells
Uploaded: 2015-03-03T14:00:00
Description: Watch this Scientific Journal Video about Imaging Local Ca2+ Signals in Cultured Mammalian Cells at JoVE.com

This work was supported by National Institutes of Health grants GM 100201 to I.F.S, and GM 048071 and GM 065830 to I.P.

We present detailed procedures for imaging local Ca2+ events in human neuroblastoma SH-SY5Y cells. These procedures can be adapted to image intracellular Ca2+ signals in many cell types8-10.
1. Preparation of Cells
<ol>
	<li>Culture the cells to be imaged according to instructions listed on their supplier&#8217;s website.</li>
	<li>A few days prior to imaging, harvest cells grown in a tissue culture flask using 1 ml of 0.25% Trypsin/EDTA (2-3 min). Following ...

<ol>
	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1 (1), 11-21 (2000).</li><li>Hill-Eubanks, D. C., Werner, M. E., Heppner, T. J., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+signaling+in+smooth+muscle.">Calcium signaling in smooth muscle.</a> Cold Spring Harb Perspect Biol. 3 (9), a004549 (2011).</li><li>Hagenston, A. 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P., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+cytosolic+calcium+based+on+rhodamine+and+fluorescein+chromophores.">Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores.</a> J Biol Chem. 264 (14), 8171-8178 (1989).</li><li>Demuro, A., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+activity+and+localization+of+single+voltage-gated+Ca(2+)+channels+by+total+internal+reflection+fluorescence+microscopy.">Imaging the activity and localization of single voltage-gated Ca(2+) channels by total internal reflection fluorescence microscopy.</a> Biophys J. 86 (5), 3250-3259 (2004).</li><li>Smith, I. F., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+quantal+substructure+of+single+inositol+trisphosphate+receptor+channel+activity+during+calcium+puffs+in+intact+mammalian+cells.">Imaging the quantal substructure of single inositol trisphosphate receptor channel activity during calcium puffs in intact mammalian cells.</a> Proc Natl Acad Sci U S A. 106 (15), 6404-6409 (2009).</li><li>Reissner, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+postsynaptic+mechanism+for+heterosynaptic+sharing+of+short-term+plasticity.">A novel postsynaptic mechanism for heterosynaptic sharing of short-term plasticity.</a> J Neurosci. 30 (26), 8797-8806 (2010).</li><li>Ellefson, K. 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We describe here protocols for imaging local Ca2+ events in cultured mammalian cells using fluorescent Ca2+ indicators. Furthermore, we describe an algorithm with an intuitive user interface that automates identification and analysis of acquired data. The procedure described here utilize the fluorescent Ca2+ indicator Cal-520, but many other Ca2+ sensitive dyes such as Fluo-3, Fluo-4, Fluo-8 and Oregon Green BAPTA-1 perform sufficiently well to image Ca2+ microdomain...

Calcium ions (Ca2+) ubiquitously regulate a diverse range of biological processes, including gene expression, secretion and long-lasting changes in synaptic plasticity1. One way through which Ca2+ can act in such a diverse manner is through the different spatial and temporal patterns of Ca2+ signals a cell can generate. For example global elevations in cytosolic [Ca2+] trigger contraction in smooth muscle tissue2 whereas smaller, localized transient elevations (local Ca2+ microdomains) stimulate gene expression essential for learning and memory3.
Free cytosolic [Ca2+] is maintained at ~100 nM at rest, but can rapidly rise to several micro-molar following the influx of Ca2+ into the cytosol through Ca2+-permeable ion channels located in the plasma membrane and by the liberation of Ca2+ from intracellular stores. Our lab focuses on the inositol 1,4,5-trisphosphate receptor (IP3R), which forms a Ca2+ release channel located in the endoplasmic reticulum (ER) membrane. Upon binding of both IP3 and Ca2+ to the cytosolic activating sites of the receptor, the IP3R channel opens to liberate Ca2+ sequestered within the ER lumen. The release of Ca2+ may remain spatially restricted to a small cluster of IP3Rs to generate a local cytosolic microdomain of Ca2+ (Ca2+ puff4) or, depending on the proximity of neighboring clusters of IP3Rs, may propagate throughout a cell by recruiting multiple puff sites through a process of Ca2+-induced Ca2+-release (CICR)5,6.
The introduction of fluorescent small molecule Ca2+ indicator dyes developed by Roger Tsien7, coupled with advanced microscopy imaging techniques, has greatly facilitated our understanding of Ca2+ signaling. Recent advances in cameras used for microscopy now allow for imaging transient local Ca2+ events such as puffs with unprecedented spatial and temporal resolution. Currently available EMCCD cameras enable imaging with 128 x 128 pixels at &#62;500 frames sec-1 (fps) and the new generation of complementary metal-oxide semiconductor (CMOS) cameras provide higher pixel resolution, and even faster speed at the expense of slightly higher noise levels. In conjunction with total internal reflection (TIRF) microscopy it is now possible to image single Ca2+ channel events8,9. This approach allows for the imaging of hundreds of channels/events simultaneously, while generating large data sets (ca. 1Gb per min) that render manual processing, visual identification and analysis impracticable and place an onus on the development of automated algorithms.
Here, we present procedures and protocols for imaging local Ca2+ signals in intact mammalian cells using fluorescent Ca2+ indicators. We further demonstrate an algorithm developed in the open-source environment Python that automates identification and analysis of local Ca2+ events imaged by both TIRF and conventional wide field epi-fluorescence (WF) microscopy. Although we describe these approaches in the context of IP3-generated Ca2+ signals, they are readily amenable to study local changes in cytosolic [Ca2+] emanating from a variety of Ca2+-permeable ion channels located in either the surface membrane or intracellular organelles8-10.

<table border="0" cellpadding="0" cellspacing="0" width="1055">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:813px;">Name</td>
			<td style="width:124px;">Company</td>
			<td style="width:119px;">Catalogue No.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Neuroblastoma SH-SY5Y cells</td>
			<td>ATCC</td>
			<td>CRL-2266</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cal-520/AM</td>
			<td>AAT Bioquest Inc.</td>
			<td>21130</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">ci-IP3 (D-23-O-Isopropylidene-6-O-(2-nitro-4.5-dimethoxy)benzyl-myo-Inositol 145-trisphosphate-Hexakis(propionoxymethyl)</td>
			<td>SiChem</td>
			<td>cag-iso-2-145-10</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO/20% pluronic F127</td>
			<td>Invitrogen</td>
			<td>P-3000MP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA/AM</td>
			<td>Invitrogen</td>
			<td>E-1219</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm glass-bottom imaging dishes</td>
			<td>MatTek</td>
			<td>P35-1.5-14-C</td>
		</tr>
	</tbody>
</table>

imaging local ca2+ signals in cultured mammalian cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, electrical excitability and cell proliferation. The diversity and specificity of Ca2+ signaling derives from mechanisms by which Ca2+ signals are generated to act over different time and spatial scales, ranging from cell-wide oscillations and waves occurring over the periods of minutes to local transient Ca2+ microdomains (Ca2+ puffs) lasting milliseconds. Recent advances in electron multiplied CCD (EMCCD) cameras now allow for imaging of local Ca2+ signals with a 128 x 128 pixel spatial resolution at rates of &#62;500 frames sec-1 (fps). This approach is highly parallel and enables the simultaneous monitoring of hundreds of channels or puff sites in a single experiment. However, the vast amounts of data generated (ca. 1 Gb per min) render visual identification and analysis of local Ca2+ events impracticable. Here we describe and demonstrate the procedures for the acquisition, detection, and analysis of local IP3-mediated Ca2+ signals in intact mammalian cells loaded with Ca2+ indicators using both wide-field epi-fluorescence (WF) and total internal reflection fluorescence (TIRF) microscopy. Furthermore, we describe an algorithm developed within the open-source software environment Python that automates the identification and analysis of these local Ca2+ signals. The algorithm localizes sites of Ca2+ release with sub-pixel resolution; allows user review of data; and outputs time sequences of fluorescence ratio signals together with amplitude and kinetic data in an Excel-compatible table.

Figure 1A shows a WF image of resting Cal-520 fluorescence in human neuroblastoma SH-SY5Y cells also loaded with ci-IP3. Exposure of these cells to a 100 msec UV flash to photo-release i-IP3 elicited transient Ca2+ puffs at discrete sites (noted by the white circles in Figure 1A). Fluorescence traces measured at these sites showed a rapid rising phase owing to transient openings of IP3Rs, followed by a much slower falling phase (Figure ...

Watch this Scientific Journal Video about Imaging Local Ca2+ Signals in Cultured Mammalian Cells at JoVE.com

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Here we present techniques for imaging local IP3-mediated Ca2+ events using fluorescence microscopy in intact mammalian cells loaded with Ca2+ indicators together with an algorithm that automates identification and analysis of these events.

Imaging Local Ca2+ Signals in Cultured Mammalian Cells

Cytosolic Ca2+ ions regulate numerous aspects of cellular activity in almost all cell types, controlling processes as wide-ranging as gene transcription, ...

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