Department of Physiology and Biophysics, University of California, Irvine (UCI)
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Beeton, C., Chandy, K. G. Induction and Monitoring of Active Delayed Type Hypersensitivity (DTH) in Rats. J. Vis. Exp. (6), e237, doi:10.3791/237 (2007).
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.
1. Preparation of the emulsion
2. Immunization of the rats
3. Challenge with antigen in the back
4. Measurement of DTH
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.
The authors are co-founders and consultants for Airmid Inc.
|Complete Freund’s adjuvant H37Ra||Reagent||Difco Laboratories||231131||Distributed by Fisher under catalog # DF 311-360-5|
|Keyhole Limpet hemocyanin||Reagent||Sigma-Aldrich||H-5654|
|5 ml glass syringes||Tool||Fisher Scientific||14-823-10 B||Need two 5 ml syringes for each batch of 5 ml of emulsion|
|18G metal bridge||Tool||Fisher Scientific||14-825-17L|
|Lewis rats||Animal||Harlan Laboratories||Female rats 7-9 weeks old|
|23G 1" needles||Tool||Fisher Scientific||14-826 AA||It is best to change needles between rats|
|3 ml Luer Lock syringes||Tool||Fisher Scientific||05-561-63|
|DPBS||Reagent||GIBCO, by Life Technologies||14040-182|
|1 ml regular tip syringes||Tool||Fisher Scientific||22-257-154|
|27G 1/2" needles||Tool||Fisher Scientific||14-826-48|
|Electric cordless trimmer||Tool||Braintree Scientific, Inc.||CLP-32130||To shave the rats before antigen challenge in the back.|
|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.|
1. Beeton, C., Chandy, K.G. Induction and monitoring of adoptive delayed-type hypersensitivity in rats (10/01/2007) Jounal of Visualized Experiments, 8, http://www.jove.com/index/Details.stp?ID=325