Name: induction and monitoring of adoptive delayed-type hypersensitivity in rats
Uploaded: 2007-10-01T00:00:00
Description: Watch this Scientific Journal Video about Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats at JoVE.com

<p class="jove_content">We thank Dr. Alexander Flugel (Martinsried, Germany) for providing us with the GFP-labeled, ovalbumin-specific T cell line used in this protocol.</p>

<p class="jove_title">1. Stimulation of the ovalbumin-specific T lymphocytes</p>
<p class="jove_content">Resting ovalbumin-specific T cells are stimulated with ovalbumin in the presence of irradiated Lewis rat thymocytes (30 Gy) as antigen presenting cells (APCs), in Stimulation Medium (Prepare in DMEM + Pen/Strep/L-Glut + NEAA + RPMI Vitamins + 2ME + Rat Serum + Ovalbumin, at the final concentrations noted in the materials list.)</p>
<p class="jove_step">Rat serum preparation</p>
<ol>
<li>We take serum from the rats we sacrifice as thymus dono...

<ol>
	<li>Beeton, C., Barbaria, J., Devaux, J., Benoliel, A. -. M., Gola, M., Sabatier, J. -. M., Bernard, D., Crest, M., Beraud, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+blocking+of+voltage-gated+K++channels+treats+experimental+autoimmune+encephalomyelitis+and+inhibits+T-cell+activation.">Selective blocking of voltage-gated K+ channels treats experimental autoimmune encephalomyelitis and inhibits T-cell activation.</a> <em style='font-style: italic'>J. Immunol</em>. , 166-936 (2001).</li><li>Beeton, C., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+monitoring+of+active+delayed+type+hypersensitivity+(DTH)in+rats.">Induction and monitoring of active delayed type hypersensitivity (DTH)in rats.</a> <em style='font-style: italic'>J Vis Exp</em>.  (5), (2007).</li></ol>

<p class="jove_content">The antigenic challenge can also be performed at other sites, for example in the skin of the back (2). We however find that challenging. Using the pinna of the ear, as shown in this video, allows for the most precise measurement of the DTH reaction.</p>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco/Invitrogen</td>
<td>12430-062</td>
<td>500ml</td>
</tr>
<tr>
<td>Sodium pyruvate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S8636</td>
<td>1 mM final, in Stimulation medium</td>
</tr>
<tr>
<td>Penicillin &#x2013; Streptomycin &#x2013; L-Glutamine</td>
<td>&nbsp;</td>
<td>Cambrex/BioWhittaker</td>
<td>17-718R</td>
<td>100 u/ml - 100 &micro;g/ml - 4 mM respectively, final, in Stimulation Medium </td>
</tr>
<tr>
<td>Non essential amino acids</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7131</td>
<td>NEAA, 1% final, in Stimulation Medium</td>
</tr>
<tr>
<td>RPMI vitamins</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7256</td>
<td>1% final, in Stimulation medium</td>
</tr>
<tr>
<td>beta-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M6250</td>
<td>2ME, 50 &micro;M final, in Stimulation Medium</td>
</tr>
<tr>
<td>Syngeneic rat serum</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1%  final. See explanation in Protocol.</td>
</tr>
<tr>
<td>Ovalbumin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5503</td>
<td>10 &micro;g/ml final, in Stimulation Medium.</td>
</tr>		</tbody>
		</table>
		</div>
	

induction and monitoring of adoptive delayed-type hypersensitivity in rats

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of an antigen against which the immune system has been primed. The inflammatory reaction is characterized by redness and swelling of the site of antigenic challenge. It is a convenient model to determine the in vivo efficacy of immunosuppressants. Cutaneous DTH can be induced either by adoptive transfer of antigen-specific T lymphocytes or by active immunization with an antigen, and subsequent intradermal challenge with the antigen to induce the inflammatory reaction in a given skin area. DTH responses can be induced to various antigens, for example ovalbumin, tuberculin, tetanus toxoid, or keyhole limpet hemocyanin. Such reactions can also be induced against autoantigen, for example to myelin basic protein (MBP) in rats with experimental autoimmune encephalomyelitis induced with MBP, an animal model for multiple sclerosis (1).
Here we demonstrate how to induce an adoptive DTH reaction in Lewis rats. We will first stimulate ovalbumin-specific T cells in vitro and inject these activated cells intraperitoneally to naive rats. After allowing the cells to equilibrate in vivo for 2 days, we will challenge the rats with ovalbumin in the pinna of one ear, while the other ear wil receive saline. The inflammatory reaction will be visible 3-72 hours later and ear thickness will be measured as an indication of DTH severity.

Watch this Scientific Journal Video about Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats at JoVE.com

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

<p class="jove_content">Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T (TEM) lymphocytes. Here we demonstrate how to activate antigen-specific TEM cells, induce adoptive DTH in Lewis rats and monitor the inflammatory response.</p>

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes that infiltrate the site of injection of ...

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