We thank Drs. John Hancock, Ray Grill, Andrew Morris and Olga Chumakova for their support. The FlexStation 3 system is part of the UTHSC Cytodynamic Imaging Facility. We also thank Drs. Ardem Patapoutian and Michael Bandell from the Scripps Research Institute and Genomics Institute of the Novartis Research Foundation (GNF) for providing the HEK293-hTRPA1 stable cell line. The research is supported in part by grants from NIH (GM081658 to MXZ) and Texas Medical Center Digestive Diseases Center (to HH) and Mission Connect/TIRR Foundation (to HH).

1. Cell preparations in 96-well plates
For HEK293 cells transiently trasfected with human TRPA1 cDNA
<ol>
<li>HEK293 cells are grown for one to two days in Dulbecco's Minimal Essential Medium (DMEM) containing 4.5 mg/ml glucose (Thermo Scientific, SH3002201), supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, 16000-044), 50 units/ml penicillin and 50 &mu;g/ml streptomycin (Invitrogen, 15140-163). On the day of the transfection, the cell density should reach 70-90...

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+calcium+channels+mediate+neurotransmitter+release+from+peripheral+neurons.">Multiple calcium channels mediate neurotransmitter release from peripheral neurons.</a> Proc Natl Acad Sci. 83, 6656-6659 (1986).</li><li>Mason, S. A., MacLeod, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+action+potential+duration+and+calcium+regulation+in+males+and+females.">Cardiac action potential duration and calcium regulation in males and females.</a> Biochem Biophys Res Commun. 388, 565-570 (2009).</li><li>Raat, N. J., Wetzels, G. E., DeMey, J. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TRPM7+regulates+cell+adhesion+by+controlling+the+calcium-dependent+protease+calpain.">TRPM7 regulates cell adhesion by controlling the calcium-dependent protease calpain.</a> J Biol Chem. 281, 11260-11270 (2006).</li><li>Dubinsky, J. M., Rothman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+concentrations+during+"chemical+hypoxia"+and+excitotoxic+neuronal+injury.">Intracellular calcium concentrations during "chemical hypoxia" and excitotoxic neuronal injury.</a> J Neurosci. 11, 2545-2551 (1991).</li><li>George, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sodium+channel+activation+augments+NMDA+receptor+function+and+promotes+neurite+outgrowth+in+immature+cerebrocortical+neurons.">Sodium channel activation augments NMDA receptor function and promotes neurite outgrowth in immature cerebrocortical neurons.</a> J Neurosci. 29, 3288-3301 (2009).</li><li>Price, K. L., Lummis, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FlexStation+examination+of+5-HT3+receptor+function+using+Ca2++-+and+membrane+potential-sensitive+dyes:+advantages+and+potential+problems.">FlexStation examination of 5-HT3 receptor function using Ca2+ - and membrane potential-sensitive dyes: advantages and potential problems.</a> J Neurosci Methods. 149, 172-177 (2005).</li><li>Laude, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+functional+assays+of+recombinant+IP3+receptors.">Rapid functional assays of recombinant IP3 receptors.</a> Cell Calcium. 38, 45-51 (2005).</li><li>Marincsak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analgesic+drug,+tramadol,+acts+as+an+agonist+of+the+transient+receptor+potential+vanilloid-1.">The analgesic drug, tramadol, acts as an agonist of the transient receptor potential vanilloid-1.</a> Anesth Analg. 106, 1890-1896 (2008).</li><li>Xie, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+high+throughput+screening+assays+against+three+subtypes+of+Ca(v)3+T-type+channels+using+molecular+and+pharmacologic+approaches.+Assay+Drug+Dev+Technol.">Validation of high throughput screening assays against three subtypes of Ca(v)3 T-type channels using molecular and pharmacologic approaches. Assay Drug Dev Technol.</a> 5, 191-203 (2007).</li><li>Christensen, B. N., Kochukov, M., McNearney, T. A., Taglialatela, G., Westlund, K. 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Intracellular Ca2+ plays important roles in many physiological and pathological conditions, such as neurotransmitter release (3), cardiac action potential (4), muscle cell contraction (5), cell signal transduction (6-7), and excitotoxic cell death (8). Measurement of intracellular Ca2+ levels helps to elucidate functional changes of Ca2+-permeable channels in the plasma membrane or intracellular organelles and the contribution of Ca2+ to the above processes (9-13). The as...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
			<th>Comments (optional)</th>
		</tr>
		<tr>
			<td>FlexStation 3		</td>
			<td>Molecular Devices</td>
			<td>FLEX3</td>
			<td>&nbsp;</td>
		</tr>		
		<tr>
			<td>96-Well, FlexStation Pipet Tips</td>
			<td>Molecular Devices</td>
			<td>9000-0912</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-Well Plates</td>
			<td>Greiner Bio-one</td>
			<td>655090</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-Well Clear Flat Bottom plates		</td>
			<td>Costar</td>
			<td>3595</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DMEM		</td>
			<td>Invitrogen</td>
			<td>12430-104</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Opti-MEM		</td>
			<td>Invitrogen</td>
			<td>31985-070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000	</td>
			<td>Invitrogen</td>
			<td>	11668-019</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluo-4 AM	</td>
			<td>Invitrogen</td>
			<td>	F14202</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PluronicF-127		</td>
			<td>Invitrogen</td>
			<td>P3000MP</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Poly-L-ornithine</td>
			<td>Sigma</td>
			<td>	P36551</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>probenecid	</td>
			<td>Sigma</td>
			<td>	P8761</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine serum albumin	</td>
			<td>Sigma</td>
			<td>	A9418</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell-based calcium assay for medium to high throughput screening of trp channel functions using flexstation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

Watch this Scientific Journal Video about Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3 at JoVE.com

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

cell-based-calcium-assay-for-medium-to-high-throughput-screening-trp

Research

JoVE Journal

Bioengineering

20.9K visualizações.  The University of Texas Health Science Center at Houston. Este vídeo fornece um protocolo detalhado para o estudo do perfil farmacológico dos direitos humanos TRPA1 canais usando FlexStation 3. O protocolo abrange detalhes de preparação das células, carregando corante e operação do leitor de microplacas, FlexStation 3.