Name: Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats (Video) | JoVE Journal
Uploaded: 2007-07-19T00:00:00.000+00:00
Duration: 13 min 26 s
Description: Learn to induce active delayed type hypersensitivity in Lewis rats using KLH antigen emulsion in Freund's adjuvant. This immunology method enables precise monitoring of inflammatory responses through swelling measurements.

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1. Preparation of the emulsion
<ol>
<li>The emulsion is a 1:1 mixture of antigen (DTH can be induced to various antigens: ovalbumin, tetanus toxoid, keyhole limpet hemocyanin....) in aqueous solution added to complete Freund's adjuvant. It is very important to add the antigen to the adjuvant and not the other way round!!! </li>
<li>There is a lot of waste when making and injecting an emulsion, so always prepare 1.5 or 2 times more than what you need. </li>
<li>Dissolve antigen in saline to prepare a 2 mg/ml solution. Do not filter, as you do not want to loose the aggregates!!! </li>
<li>Put complete Freund's adjuvant H37Ra (Difco, catalog # 231131) in a tube and vortex at high speed. Add the antigen drop by drop while vortexing. When all of the antigen is added, vortex for 5 more minutes. The mixture should turn white. </li>
<li>Put the emulsion in a glass syringe and link it to another glass syringe using a 18G bridge (Fisher catalog # 14-825-17L). Send the emulsion from a syringe to the other until it becomes hard (5-10 min). It is best to use two 5 ml syringes. If more than 5 ml of emulsion are required, use several sets of 5 ml syringes. </li>
<li>The emulsion can be stored in the syringes at 4&deg;C for 3 weeks. </li>
</ol>
2. Immunization of the rats
<ol>
<li>Mix the emulsion in the syringes for a few minutes and transfer to the syringe used for the injection. Inject 200 ml subcutaneously in 1-2 sites at the base of the tail using 23G needles and a Luer-Lok tip 3 ml syringe. </li>
</ol>
3. Challenge with antigen in the back
<ol>
<li>Seven days after immunization, shave 2 spots on the backs of the rats and inject intradermally 20 ml of a 1 mg/ml antigen solution in one spot and saline (or irrelevant antigen) in the other spot. Do NOT filter the antigen solution, as you may loose aggregates that enhance the inflammatory reaction. </li>
<li>The rats should be anesthesized to avoid movements. Inject the solution intradermally using 27G &frac12; needles and 1 ml syringes. Change needles between injections. Note: the challenge can be done in the pinna of the ears instead of the back for more precise measurements. See video at <a href="https://www.jove.com/index/Details.stp?ID=325">https://www.jove.com/index/Details.stp?ID=325</a> </li>
</ol>
4. Measurement of DTH
<ol>
<li>The injection site will start turning red and swelling as a sign of inflammation. Measure the diameter of the inflamed area using a ruler or, better yet,&nbsp;a caliper. The maximum swelling should occur between 24 and 72 hours. </li>
</ol>

<ol>
	<li>Beeton, C., Chandy, K. G. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+and+monitoring+of+adoptive+delayed-type+hypersensitivity+in+rats.">Induction and monitoring of adoptive delayed-type hypersensitivity in rats.</a> Journal of Visualized Experiments. 8, (2007).</li></ol>

The authors are co-founders and consultants for Airmid Inc.

For a more precise measurement of the DTH reaction we recommend challenging the rats with the antigen in the pinna of the ear. The reaction can then be measured as a difference in ear swelling between the relevant antigen-challenged ear and the ear challenged with either saline alone or with the irrelevant antigen. A demonstration of the challenge in the ear is shown in 1.

<table><tbody><tr><td>Complete Freund&amp;rsquo;s adjuvant H37Ra</td><td>Reagent</td><td>Difco Laboratories</td><td>231131</td><td>Distributed by Fisher under catalog # DF 311-360-5</td></tr><tr><td>Keyhole Limpet hemocyanin</td><td>Reagent</td><td>Sigma-Aldrich</td><td>H-5654</td><td></td></tr><tr><td>Ovalbumin</td><td>Reagent</td><td>Sigma-Aldrich</td><td>A-5503</td><td></td></tr><tr><td>5 ml glass syringes</td><td>Tool</td><td>Fisher Scientific</td><td>14-823-10 B</td><td>Need two 5 ml syringes for each batch of 5 ml of emulsion</td></tr><tr><td>18G metal bridge</td><td>Tool</td><td>Fisher Scientific</td><td>14-825-17L</td><td></td></tr><tr><td>Lewis rats</td><td>Animal</td><td>Harlan Laboratories</td><td></td><td>Female rats 7-9 weeks old</td></tr><tr><td>23G 1&quot; needles</td><td>Tool</td><td>Fisher Scientific</td><td>14-826 AA</td><td>It is best to change needles between rats</td></tr><tr><td>3 ml Luer Lock syringes</td><td>Tool</td><td>Fisher Scientific</td><td>05-561-63</td><td></td></tr><tr><td>DPBS</td><td>Reagent</td><td>GIBCO, by Life Technologies</td><td>14040-182</td><td></td></tr><tr><td>1 ml regular tip syringes</td><td>Tool</td><td>Fisher Scientific</td><td>22-257-154</td><td></td></tr><tr><td>27G 1/2&quot; needles</td><td>Tool</td><td>Fisher Scientific</td><td>14-826-48</td><td></td></tr><tr><td>Electric cordless trimmer</td><td>Tool</td><td>Braintree Scientific, Inc.</td><td>CLP-32130</td><td>To shave the rats before antigen challenge in the back.</td></tr><tr><td colspan="5">Use sterile solutions reagents and syringes. The glass syringes and 18G bridges can be autoclaved. Read material data sheet before handling a reagent for the first time and wear appropriate protective equipment.</td></tr></tbody></table>

induction and monitoring of active delayed type hypersensitivity (dth) in rats

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) 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 (KLH).
Here we demonstrate how to induce an active DTH reaction in Lewis rats. We will first prepare a water-in-oil emulsion of KLH, our antigen of interest, in complete Freund's adjuvant and inject this emulsion subcutaneously to rats. This will prime the immune system to develop memory T cells directed to KLH. Seven days later we will challenge the rats intradermally on the back with KLH on one side and with ovalbumin, an irrelevant antigen, on the other side. The inflammatory reaction will be visible 16-72 hours later and the red and swollen area will be measured as an indication of DTH severity.

Delayed type hypersensitivity (DTH) is an inflammatory reaction mediated by CCR7- effector memory T lymphocytes. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

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

Hi, I am Christine Beaton. I work in George Chandy lab in the Department of Physiology and biophysics at UCI. Today I'm going to show you how to induce active DTH in rats.So for that I'm going to prepare an emulsion of KLH, which is my antigen in France adjuvant. And then I'm going to inject this emulsion at the base of the tail of Lewis Rats. And this is going to induce immunization of the rats against KH.And in one week I'm going to challenge the rats with KH and measure the DTH reaction by swelling measurement of the swelling.That's the site of challenge and we are using that in the lab to see the effect of PO channel blockers on inflammatory reactions such as DTH. So here is the incomplete trans adjuvant I'm going to use to make the emulsion. I could also use complete trans adjuvant.Here are quartz of KLH, which is my antigen today, and I have a solution here at two milligrams per ml. In PBS, I'm going to use this five ml syringe to take out the incomplete trans adjuvant and transfer it into a 15 mil tube. I'm then going to put a tube on the vertex and while vortexing, I'm going to add my antigen drop by drop to the adjuvant.And then I'm going to vortex for further five minutes

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JoVE Journal

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Learn to induce active delayed type hypersensitivity in Lewis rats using KLH antigen emulsion in Freund's adjuvant. This immunology method enables precise monitoring of inflammatory responses through swelling measurements.

Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats

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