This work was supported by grants from the National Institutes of Health (ES07408 and EB00208).

1. ECIS/Taxis Electrode Preparation
<ol>
 <li>The gold surface of the ECIS/taxis electrode array (consisting of 8 chambers per slide) is first stabilized by pre-treatment with sterile 10 mM cysteine in deionized water (dH2O) for 15 min at room temperature under sterile conditions.</li>
 <li>Aspirate the cysteine solution from each electrode chamber, rinse 3 times with sterile dH2O, and replace with 250 &mu;l of complete medium (RPMI 1640, 10%FBS, 25 mM HEPES buffer).</li>
 <li>Connect the electrod...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+of+actin+filaments+by+myosin+II+is+a+major+contributor+to+cortical+integrity+and+cell+motility+in+restrictive+environments.">Cross-linking of actin filaments by myosin II is a major contributor to cortical integrity and cell motility in restrictive environments.</a> J. Cell. Sci. 116, 3761-3770 (2003).</li><li>Condeelis, J., Bresnick, A., Demma, M., Dharmawardhane, S., Eddy, R., Hall, A. L., Sauterer, R., Warren, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+amoeboid+chemotaxis:+an+evaluation+of+the+cortical+expansion.">Mechanisms of amoeboid chemotaxis: an evaluation of the cortical expansion.</a> 11, 5-6 (1990).</li><li>Hadjout, N., Laevsky, G., Knecht, D. A., Lynes, M. 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David A Knecht and Michael A Lynes have an issued patent for the ECIS/Taxis technology, which as been licensed by the University of Connecticut to Applied Biophysics, Inc.

Novel characteristics of the ECIS/Taxis assay include its ability to automate the collection of real-time data as cells respond to chemoattractant. While the most commonplace application of this technology is to measure cellular responses to individual chemotactic gradients, or to gradients comprised of mixtures of chemotaxis agonists and antagonists, the ECIS/Taxis approach is also amenable to variations to these configurations that could be quite helpful in the assessment of cellular responsiveness. There is good evide...

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog number </td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>ECIS Z&theta;</td>
			<td>Applied Biophysics</td>
			<td>&nbsp;</td>
			<td><a href="http://www.biophysics.com/prodducts_Ecisz0.php">http://www.biophysics.com/prodducts_Ecisz0.php</a></td>
		</tr>
		<tr>
			<td>ECIS Electrode Array</td>
			<td>Applied Biophysics</td>
			<td>8W Chemotaxis</td>
			<td><a href="http://www.biophysics.com/cultureware.php">http://www.biophysics.com/cultureware.php</a></td>
		</tr>
		<tr>
			<td>Seakem GTG agarose</td>
			<td>BioWhittaker Molecular Applications</td>
			<td>50070</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Cellgro</td>
			<td>10-040</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HyClone Fetal Bovine Serum</td>
			<td>Thermo Scientific</td>
			<td>SH300703</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>MP Biomedicals</td>
			<td>1670049</td>
			<td>Penicillin 5,000 IU/ml; Streptomycin 5 mg/ml</td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>MP Biomedicals</td>
			<td>1688449</td>
			<td>1M solution, cell culture grade</td>
		</tr>
		<tr>
			<td>14 Gauge stainless steel Cannula (2) 4 inch</td>
			<td>General Laboratory Supply</td>
			<td>5-8365-1</td>
			<td>Blunt point</td>
		</tr>
	</tbody>
</table>

measurement of cellular chemotaxis with ecis/taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

Watch this Scientific Journal Video about Measurement of Cellular Chemotaxis with ECIS/Taxis at JoVE.com

Measurement of Cellular Chemotaxis with ECIS/Taxis

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

measurement-of-cellular-chemotaxis-with-ecistaxis

Research

JoVE Journal

Biology

14.7K visualizações.  University of Connecticut. O sistema ECIS / Taxis é um sistema automatizado, o ensaio em tempo real que mede a quimiotaxia celulares. Neste ensaio, as células se movem por baixo de uma camada de agarose para se chegar a um eléctrodo de destino. Movimento celular é medida pelo aparecimento de resistência a AC 0 actual.