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://jove.unicartagenaproxy.elogim.com/index/Details.stp?ID=325">https://jove.unicartagenaproxy.elogim.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="http://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.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<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>Complete Freund's adjuvant H37Ra</td>
<td>Reagent</td>
<td>Difco</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</td>
<td>H-5654</td>
<td>&nbsp;</td>
<tr>
<td>Ovalbumin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5503</td>
<td>&nbsp;</td>
<tr>
<td>5 ml glass syringes</td>
<td>Tool</td>
<td>Fisher</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</td>
<td>14-825-17L</td>
<td>&nbsp;</td>
<tr>
<td>Lewis rats</td>
<td>Animal</td>
<td>Harlan Sprague Dawley</td>
<td>&nbsp;</td>
<td>Female rats 7-9 weeks old</td>
</tr>
<tr>
<td>23G 1" needles</td>
<td>Tool</td>
<td>Fisher</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</td>
<td>05-561-63</td>
<td>&nbsp;</td>
<tr>
<td>DPBS</td>
<td>Reagent</td>
<td>Gibco/Invitrogen</td>
<td>14040-182</td>
<td>&nbsp;</td>
<tr>
<td>1 ml regular tip syringes</td>
<td>Tool</td>
<td>Fisher</td>
<td>22-257-154</td>
<td>&nbsp;</td>
<tr>
<td>27G 1/2" needles</td>
<td>Tool</td>
<td>Fisher</td>
<td>14-826-48</td>
<td>&nbsp;</td>
<tr>
<td>Electric cordless trimmer</td>
<td>Tool</td>
<td>Braintree Scientific</td>
<td>CLP-32130</td>
<td>To shave the rats before antigen challenge in the back.</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
			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.		</div>

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.

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

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. Here we demonstrate how to induce active DTH in Lewis rats and monitor the inflammatory response.

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 ...

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Universidad de Cartagena UDEC

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JoVE Journal

Biology

17.7K Views.  University of California, Irvine (UCI). 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.

Video: Induction and Monitoring of Active Delayed Type Hypersensitivity DTH in Rats

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.

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.

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a method for inducing and recording the progress of a DTH reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the CCR7- effector / memory T cell response.
An adoptive DTH is induced by the intraperitoneal injection of GFP-labeled Ova-specific CCR7- effector / memory T cell line (Beeton, C J. Visualized Experiments, Issue 8). Cells are then allowed to equilibrate in the rat for 48 hours before challenge by injecting one ear with saline (control ear) and the other with a 1:1 mix of Ova and Ova conjugated to Texas-Red (Ova-TR) to allow visualization of resident antigen-presenting cells.
We describe a method of tissue preparation useful for imaging the motility of cells within the deep dermal layer during an immune response, in conjunction with visualization of collagen fibers by second harmonic generation. Ear tissue is cut into 5 x 5 mm squares (slightly larger is better) and mounted onto plastic cover slips using Vetbond&trade;, which are then secured using silicone grease in an imaging chamber and superfused by oxygen-bubbled tissue culture medium at 37&deg;C.

Here we demonstrate a method for inducing and recording the progress of a delayed type-hypersensitivity (DTH) reaction in the rat ear. This is followed by a demonstration of the preparation of rat ear tissue for two-photon imaging of the effector / memory T cell response.

Delayed type hypersensitivity (DTH) is an immune reaction in which the main players are CCR7- effector / memory T lymphocytes. Here, we demonstrate a ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

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Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

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Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

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Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

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Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Chromatographic Purification of Highly Active Yeast Ribosomes

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Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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

Summary

Explore More Videos

Chapters in this video

Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats

Imaging Effector Memory T cells in the Ear After Induction of Adoptive DTH

Assay for Neural Induction in the Chick Embryo

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

In vitro Reconstitution of the Active T. castaneum Telomerase

Induction and Testing of Hypoxia in Cell Culture

Chromatographic Purification of Highly Active Yeast Ribosomes

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory