This study was supported by subvention SUBZ.A020.22.060 of the Medical University in Wroclaw, Poland, and by grants from the Ministry of Science and Higher Education N N401 545940 to MS and IP2012 0443 72 to MMS.

<ol>
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Kinetics of fibrinogen/fibrin accumulation and vascular permeability changes in tuberculin and cutaneous basophil hypersensitivity reactions.</a> Journal of Immunology. 114, 377-387 (1975).</li><li>Szczepanik, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+contact+sensitivity+in+non-obese+diabetic+(NOD)+mice+by+innate+immunity.">Regulation of contact sensitivity in non-obese diabetic (NOD) mice by innate immunity.</a> Contact Dermatitis. 79 (4), 197-207 (2018).</li><li>Askenase, P. W., Majewska-Szczepanik, M., Kerfoot, S., Szczepanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Participation+of+iNKT+cells+in+the+early+and+late+components+of+Tc1-mediated+DNFB+contact+sensitivity:+Cooperative+role+of+&#947;&#948;-T+cells.">Participation of iNKT cells in the early and late components of Tc1-mediated DNFB contact sensitivity: Cooperative role of &#947;&#948;-T cells.</a> Scandinavian Journal of Immunology. 73 (5), 465-477 (2011).</li><li>Zemelka-Wi&#261;cek, M., Majewska-Szczepanik, M., Ptak, W., Szczepanik, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epicutaneous+immunization+with+protein+antigen+induces+antigen-non-specific+suppression+of+CD8+T+cell+mediated+contact+sensitivity.">Epicutaneous immunization with protein antigen induces antigen-non-specific suppression of CD8 T cell mediated contact sensitivity.</a> Pharmacological Reports. 64 (6), 1485-1496 (2012).</li><li>Van Loveren, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+micrometers+and+calipers+to+measure+various+components+of+delayed-type+hypersensitivity+ear+swelling+reactions+in+mice.">Use of micrometers and calipers to measure various components of delayed-type hypersensitivity ear swelling reactions in mice.</a> Journal of Immunological Methods. 67 (2), 311-319 (1984).</li><li>Tsuji, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell-dependent+T+cell+responses:+IgM+antibodies+are+required+to+elicit+contact+sensitivity.">B cell-dependent T cell responses: IgM antibodies are required to elicit contact sensitivity.</a> The Journal of Experimental Medicine. 196 (10), 1277-1290 (2002).</li><li>Campos, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutaneous+immunization+rapidly+activates+liver+invariant+Valpha14+NKT+cells+stimulating+B-1+B+cells+to+initiate+T+cell+recruitment+for+elicitation+of+contact+sensitivity.">Cutaneous immunization rapidly activates liver invariant Valpha14 NKT cells stimulating B-1 B cells to initiate T cell recruitment for elicitation of contact sensitivity.</a> The Journal of Experimental Medicine. 198 (12), 1785-1796 (2003).</li><li>R&#252;hl-Muth, A. C., Maler, M. D., Esser, P. R., Martin, S. 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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+hypersensitivity+in+MHC+class+II-deficient+mice+depends+on+CD8+T+lymphocytes+primed+by+immunostimulating+Langerhans+cells.">Contact hypersensitivity in MHC class II-deficient mice depends on CD8 T lymphocytes primed by immunostimulating Langerhans cells.</a> The Journal of Investigative Dermatology. 111 (1), 44-49 (1998).</li><li>Martin, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+immunization+indicates+that+CD8++T+cells+are+the+dominant+effector+cells+in+trinitrophenyl-specific+contact+hypersensitivity.">Peptide immunization indicates that CD8+ T cells are the dominant effector cells in trinitrophenyl-specific contact hypersensitivity.</a> The Journal of Investigative Dermatology. 115 (2), 260-266 (2000).</li><li>Vennegaard, M. 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The authors have nothing to disclose.

CHS is induced via haptens, which bind to self-protein antigens in the skin, creating neoantigens. CHS is mediated by local extravascular recruitment of circulating antigen-specific CHS-effector T cells, which results in swelling in the challenged tissue, peaking 24 h after exposure of the secondary skin to the same hapten6. The swelling of the tissue is mainly caused by the infiltration of leukocytes and leukocyte-dependent fibrin deposition24. These changes can be detected with a micrometer measuring the ear swelling of hapten-sensitized and challenged versus sham-sensitized and challenged mice.
CHS can also be determined by the comparison of ear weight. Then, the ear punches utilized for ear weight determination can be used for further tests. The cell infiltrates in the inflamed ears consist of cytokine-producing T lymphocytes and MPO-positive neutrophils. In the ear tissue homogenates, various parameters can be measured, such as MPO activity or the concentration of pro-inflammatory cytokines (e.g., TNF-&#945;, IFN-&#947;, IL-17A, or others) using the ELISA test or the expression of the cytokine mRNA in the skin using the qPCR test25. Additionally, vessel permeability changes can be evaluated using the Evans blue test21,26.
In the inflamed ear tissue, the changes observed can also be complemented with in vitro tests, highlighting T cell proliferation and cytokine production. This can be readily accomplished by culturing ALNs in the presence of hapten-conjugated protein antigens22,27. The evaluation of cytokine secretion by ALNs isolated from sensitized but not challenged mice provides details regarding cytokine production by the CHS-effector cells in the place of their induction.
Limitations 
Many micrometers measure ear swelling with differing accuracy. For example, the lowest pressure is exerted by a spring caliper, so it seems that the results will best reflect the actual thickness ear measurement. However, micrometers that exert more pressure, such as Mitutoyo, are likely to compress more tightly the fluid that accumulates in the ears in the early phase of edema formation. The biphasic nature of CHS can be visualized more clearly using such micrometers because more pressure is exerted. This is more difficult when using a spring caliper with light pressure28. Nevertheless, in some studies, the thickness of the ears was measured with a caliper29. Also, the observer&#39;s experience ensures accurate measurements, which can be influenced by subjective feelings, even if the observer is unaware of the experimental groups.
Alternative methods have been described here that can help confirm the measurements of ear swelling with a micrometer, making the data presented more reliable and less subjective. However, these alternative strategies for assessing CHS can only be used at one point in time. Micrometer measurements can be repeated at various time points, allowing for studying the CHS kinetics.
Modifications 
The protocol for studying the CHS reaction we use in our laboratory differs significantly from those used in other laboratories, including the dose of hapten and the solvent composition used for both elicitation and sensitization, as well as the time point at which the reaction is assessed. The ear thickness can be measured at different time points (e.g., 2 h, 24 h, 48 h, and 72 h after the challenge)10,30. Table 1 shows different experimental models, particularly the differences in the animal strain, hapten, and the sensitization/challenge times used in various studies10,21,22,26,31,32,33,34,35,36,37. The CHS protocol might also be conducted without previous shaving of the abdomen of the mice.
The next difference concerns the execution of the elicitation (challenge) itself. As is typical, the sensitization is done by painting shaved mice&#39;s abdomen skin with the hapten or vehicle alone. Subsequently, in both groups, one ear is challenged with diluted hapten on each ear side. As a control, the opposite ear is painted with an identical amount of vehicle alone10,31.
Critical steps 
The most critical moment is to trigger CHS, as the test results depend on it.&#160;(1) Hapten solution is very volatile and light sensitive, so it must be tightly closed and protected from light, and when in use, it must be quickly applied to the skin of the animals so it will not evaporate. (2) After applying the hapten to the skin of the abdomen, it must be dried before the animal returns to the cage because mice can smear it against the bedding or apply it to the ears with their paws (then the measurement of the thickness of the auricle may be inadequate). (3) Various methods of labeling animals, such as marking mouse tails with a solvent-resistant marker, are also known. However, the dyes present in the marker might (not studied) become a hapten and trigger CHS. Therefore, in this study, a marking method was chosen that does not affect the reaction. (4) Choosing a shaving method is very important as it can irritate the skin. In this protocol, a gray soap was used because it has soothing properties; it accelerates the treatment of minor cuts and festering wounds, which is helped by its antibacterial effects. It soothes swelling and inflammation of the skin.
Further investigations and standardization of the experimental procedures, including the animal use (strain and sex), are required to compare the results from different studies.
Many different environmental factors and new substances with biological functions can be tested in the CHS model. This model can be useful if researchers want to show if tested factors modulate T cell-dependent immune responses.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/64329">Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis</a>. The Protocol&#160;and Representative Results sections were updated.
Step 3.2.1 of the Protocol has been updated from:
On day &#34;4&#34;, prepare 0.4% hapten: TNCB in an acetone-ethanol mixture (ratio 1:1). Prepare the solution just before use in a glass vial and protect it from light by covering the vial with aluminum foil.
to:
On day &#34;4&#34;, prepare 0.4% hapten: TNCB in an acetone: olive oil&#160;mixture (ratio 1:1). Prepare the solution just before use in a glass vial and protect it from light by covering the vial with aluminum foil.
Figure 1 of the Representative Results has been updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64329/64329fig01.jpg" /> 
Figure 1: Induction of CHS. Sensitization, challenge, and ear measurement. Abbreviations: CHS = contact hypersensitivity reaction; TNCB = 2,4,6-trinitrochlorobenzene. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64329/64329fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64329/64329fig1v2.jpg" /> 
Figure 1: Induction of CHS. Sensitization, challenge, and ear measurement. Abbreviations: CHS = contact hypersensitivity reaction; TNCB = 2,4,6-trinitrochlorobenzene. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64329/64329fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 2 of the Representative Results has been updated from:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64329/64329fig02.jpg" /> 
Figure 2: Adoptive transfer of the CHS-effector cells. Abbreviations: ALNs =axillary and inguinal lymph nodes; CHS = contact hypersensitivity reaction; i.v. = intravenously; SPLs = spleens; TNCB = 2,4,6-trinitrochlorobenzene. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64329/64329fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64329/64329fig2v2.jpg" /> 
Figure 2: Adoptive transfer of the CHS-effector cells. Abbreviations: ALNs =axillary and inguinal lymph nodes; CHS = contact hypersensitivity reaction; i.v. = intravenously; SPLs = spleens; TNCB = 2,4,6-trinitrochlorobenzene. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64329/64329fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/t/64329">Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis</a>. The Protocol&#160;and Representative Results sections were updated.

Erratum: Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Allergic contact dermatitis (ACD) is a common skin inflammatory disease in industrialized countries caused by a type IV hypersensitivity reaction resulting from exposure to low molecular weight chemicals called haptens. The substances causing contact sensitization in humans include heavy metal ions (chromium, nickel, iron, cobalt), turpentine, fragrances, dyes, and preservatives present in cosmetics (e.g., p-phenylenediamine), some drugs (e.g., neomycin, benzocaine), &#946;-lactam antibiotics (i.e., penicillin), chemicals produced by plants (pentadecacatechol, a substance present in poison ivy), as well as hydroquinone-used in the photographic industry1,2. ACD etiological agents are very high as over 100,000 chemicals are used in industry alone, and 2,000 new ones are synthesized each year. To date, more than 3,700 molecules have been identified that may be contact haptens/allergens3. The contact hypersensitivity reaction (CHS) is an experimental model of ACD that can be studied in mice, guinea pigs, and rats and can be induced by the topical skin application of reactive chemical haptens dissolved in organic solvents4,5,6. This study aims to describe an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests.
The CHS consists of sensitization (induction) and effector (challenge) phases. In animal models, haptens first bind covalently to proteins in the body to create neoantigens. During the sensitization phase, activated keratinocytes promote the migration and maturation of skin dendritic cells (sDCs) by producing pro-inflammatory cytokines-tumor necrosis factor &#945; (TNF-&#945;) and interleukin 1&#946; (IL-1&#946;)7. Epidermal Langerhans cells (LCs) present antigens during the CHS induction and effector phases8. LCs exposed to hapten during sensitization promote the induction of both regulatory and effector cells9. Increasing evidence from several studies suggests that CHS responses can be mediated by either CD4+ MHC class II-restricted Th1 cells, locally releasing interferon-&#947; (IFN-&#947;) to employ a characteristic inflammatory infiltrate, CD8+ MHC class I-restricted Tc1 lymphocytes that can also release IFN-&#947; but mostly mediate cytotoxic damage to keratinocytes, and now also interleukin 17 (IL-17)-producing Th17 cells10,11.
Several different CHS models employing various species12,13,14 and haptens have been developed (a detailed comparison of different haptens, solvents, and time of application is summarized in Table 1). The mouse, a frequently used laboratory species, offers a few advantages in studying CHS. There are more strains, knockouts (KO), and transgenic animals among mice compared to other species, which makes them a very attractive animal15. In addition, the CHS model requires many animals, and mice are more economical here. Animal models do not mimic ACD in all aspects; in particular, they show crusting and desquamation, which is not common for humans16,17. The features of chronic disease are challenging to reproduce, mainly because the described model does not assume the application of the hapten for a long period of time. However, it has been confirmed here that many of the significant aspects of ACD are reproduced. It has also been shown that, as in humans, these features are associated with local allergic reactions. The choice of hapten, its solvent, and its application outlined in this protocol were dictated by the fact that the results have been confirmed by numerous in vitro tests and that it was tested and modified in the laboratory for many years until the current version was established. Murine models allow for the analysis of the cell subsets or cytokines that are involved in the development of ACD and are essential for preclinical assessments of new treatments.

<table><tbody><tr><td>70% ethanol</td><td>Merck KGaA, Darmstadt, Germany</td><td>65350-M</td><td>for surface disinfection</td></tr><tr><td>96-well flat-bottom plates, polypropylene</td><td>Greiner Bio-One GmbH, Kremsmunster, Austria</td><td>655101</td><td>for MPO and Evans blue measurement - plates should be made of polypropylene, that has a lower binding capacity so proteins or DNA will not bind</td></tr><tr><td>Acetone (ACS reagent, &amp;ge;99.5%)</td><td>Merck KGaA, Darmstadt, Germany</td><td>179124</td><td></td></tr><tr><td>Aluminum foil</td><td>Merck KGaA, Darmstadt, Germany</td><td>Z185140</td><td></td></tr><tr><td>Analytical balance</td><td>Sartorius Weighing Technology GmbH, Goettingen, Germany</td><td>PRACTUM224-1s, 29105177</td><td></td></tr><tr><td>Automated tissue processor</td><td>MediMeas Instruments, Sarsehri, Haryana, India</td><td>MTP-E-212</td><td>automatically prepare tissue samples by fixing, dehydrating, clearing, and infiltrating them with paraffin</td></tr><tr><td>BD Vacutainer SST II Advance (tube with gel for obtaining serum)</td><td>Becton Dickinson (BD), Franklin Lakes, NJ, USA</td><td>BD 366882</td><td></td></tr><tr><td>Bicinchoninic acid kit for protein determination</td><td>Merck KGaA, Darmstadt, Germany</td><td>BCA1-1KT</td><td></td></tr><tr><td>Biotin Rat Anti-Mouse IgG1</td><td>Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA</td><td>553441</td><td></td></tr><tr><td>BSA (bovine serum albumine)</td><td>Merck KGaA, Darmstadt, Germany</td><td>A9418</td><td>protein assays &amp;amp; analysis, 2 mg/mL</td></tr><tr><td>Cell strainer, pore size 70 &amp;mu;m</td><td>BIOLOGIX, China</td><td>15-1070</td><td>suitable for 50 mL tubes</td></tr><tr><td>Coverslip</td><td>VWR, Radnor, Pennsylvania, United States</td><td>631-1583</td><td>24 mm, but it possible to use different size</td></tr><tr><td>Disposable pipettes capacity: 5 mL, 10 mL, 25 mL</td><td>Merck KGaA, Darmstadt, Germany</td><td>Z740301, Z740302, Z740303</td><td></td></tr><tr><td>DPBS (Dulbecco&amp;prime;s phosphate buffered saline)</td><td>ThermoFisher Scientific,&amp;nbsp; Waltham, Massachusetts, USA</td><td>14190144</td><td>no calcium, no magnesium, mammalian cell culture</td></tr><tr><td>DPX Mountant for histology</td><td>Merck KGaA, Darmstadt, Germany</td><td>6522</td><td>mounting media for H-E, might be used some other e.g, Canada balsam</td></tr><tr><td>Dumont 5 tweezers - straight</td><td>Animalab, Poznan, Poland</td><td>11251-10FST</td><td>surgical instruments for procedures on mice (should be steriled)</td></tr><tr><td>Dumont 7 tweezers - bent</td><td>Animalab, Poznan, Poland</td><td>11272-50FST</td><td>surgical instruments for procedures on mice (should be steriled)</td></tr><tr><td>Eosin Y solution, alcoholic</td><td>Merck KGaA, Darmstadt, Germany</td><td>HT110116</td><td></td></tr><tr><td>Eppendorf Safe-Lock Tubes 1.5 mL</td><td>Eppenforf, Germany</td><td>3,01,20,086</td><td>polypropylene</td></tr><tr><td>Eppendorf Safe-Lock Tubes, 2.0 mL</td><td>Eppenforf, Germany</td><td>3,01,20,094</td><td>polypropylene, round bottom (the homogenization beads can easily move)</td></tr><tr><td>Ethanol 100% (absolute alkohol)</td><td>Merck KGaA, Darmstadt, Germany</td><td>1.07017</td><td></td></tr><tr><td>Ethanol 96%</td><td>Merck KGaA, Darmstadt, Germany</td><td>1.59010</td><td></td></tr><tr><td>Evans blue</td><td>Merck KGaA, Darmstadt, Germany</td><td>E2129</td><td></td></tr><tr><td>FBS (fetal bovine serum)</td><td>ThermoFisher Scientific, Waltham, Massachusetts, USA</td><td>A3160802</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Merck KGaA, Darmstadt, Germany</td><td>HT501128</td><td></td></tr><tr><td>Formamide 99.5% (GC)</td><td>Merck KGaA, Darmstadt, Germany</td><td>F7503</td><td></td></tr><tr><td>Freezer -20 &amp;deg;C</td><td>Bosch, Germany</td><td>GSN54AW30</td><td></td></tr><tr><td>Fridge +4 &amp;deg;C / freezer -20 &amp;deg;C</td><td>Bosch, Germany</td><td>KGV36V10</td><td>mammalian Cell Culture, qualified, Brazil, 10 x 50 mL</td></tr><tr><td>Glass microskope slides, SuperFrost Plus</td><td>VWR, Radnor, Pennsylvania, United States</td><td>631-0108, 631-0446, 631-0447, 631-0448, 631-0449</td><td>Slides that eliminates the need to apply adhesive materials or protein coatings, to preventing any tissue sections loss during staining.</td></tr><tr><td>Graph Pad Prism</td><td>GraphPad Software Inc.</td><td>v. 9.4.0</td><td></td></tr><tr><td>Grey soap</td><td>Pollena Ostrzesz&amp;oacute;w, Producent Chemii Gospodarczej Sp. Z.o.o. , Sp. K., Poland</td><td>Bialy jelen soap bar</td><td>grey Soap Bar Natural Hypoallergenic. Generally available product</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt; (sulfuric acid) 1 mol/l (1 M)</td><td>Merck KGaA, Darmstadt, Germany</td><td>1.60313</td><td></td></tr><tr><td>Harris hematoxylin solution</td><td>Merck KGaA, Darmstadt, Germany</td><td>HHS16</td><td></td></tr><tr><td>Hemocytometer</td><td>VWR, Avantor, U.S.A</td><td>612-5719</td><td>manual counting chamber is recommend, which is more accurate</td></tr><tr><td>Hexadecyltrimethylammonium bromide</td><td>Merck KGaA, Darmstadt, Germany</td><td>H5882</td><td></td></tr><tr><td>Homogenizer</td><td>QIAGEN Hilden, Germany</td><td>Tissue Lyser LT, SN 23.1001/05234</td><td>homogenizer with stainless steel beads (diameter 5 mm) for 2 mL centrifuge tubes</td></tr><tr><td>Horseradish peroxidase streptavidin (HRP streptavidin)</td><td>Vector Laboratories, Inc., Burlingame, CA, USA</td><td>SA-5004-1</td><td></td></tr><tr><td>Hydrogen peroxide solution (H&lt;sub&gt;2&lt;/sub&gt;0&lt;sub&gt;2&lt;/sub&gt;)</td><td>Merck KGaA, Darmstadt, Germany</td><td>H1009</td><td></td></tr><tr><td>Incubator Heracell 150i</td><td>Thermo Electron LED Gmbh, Germany</td><td>41071068</td><td>37 &lt;sup&gt;o&lt;/sup&gt;C in the atmosphere of 5 % CO&lt;sub&gt;2&lt;/sub&gt;, and 65 &lt;sup&gt;0&lt;/sup&gt;C for deparaffinization the sections for histology</td></tr><tr><td>Ketamine 100 mg/mL, solution for injection</td><td>Biowet Pulawy Sp. z o.o., Pulawy, Poland</td><td>cat.# not avaliable</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (potassium dihydrogen phosphate) 99.995% anhydrous basis</td><td>Merck KGaA, Darmstadt, Germany</td><td>1.05108</td><td></td></tr><tr><td>Laboratory Centrifuge</td><td>Heraeus Megafuge 1.0R, Thermo Scientific, Germany</td><td>B00013899</td><td>speed to 300 x g, with cooling to 4 &lt;sup&gt;0&lt;/sup&gt;C</td></tr><tr><td>Laboratory Centrifuge</td><td>Heraeus Fresco 21, Thermo Scientific, Germany</td><td>75002425</td><td>speed to 3,000 x g, with cooling to 4 &lt;sup&gt;0&lt;/sup&gt;C</td></tr><tr><td>Mask (FFP2)</td><td>VWR, Radnor, Pennsylvania, United States</td><td>111-0917</td><td>for working with ortho-dianisine dihydrochloride</td></tr><tr><td>Mice</td><td>Breeding unit of the Chair of Biomedical Sciences, Faculty of Health Sciences, Jagiellonian University Medical College, Krakow, Poland</td><td>CBA/J, C57BL/6</td><td></td></tr><tr><td>Micrometer</td><td>Mitutoyo, Tokyo, Japan</td><td>193-111</td><td>digit Outside Micrometer, Ratchet Stop, 0-25mm Range, 0.001mm Graduation, +/-0.002mm Accuracy, https://shop.mitutoyo.pl/web/mitutoyo/pl_PL/all/all/Mikrometr%20analogowy%20/PR/193-111/index.xhtml&amp;nbsp;&amp;nbsp;</td></tr><tr><td>microplate, 96 well, microlon, high binding (for ELISA test)</td><td>Greiner Bio-One GmbH, Kremsmunster, Austria</td><td>655061</td><td>with maxi-sorp binding surfaces for reliable and reproducible results in colormetric assays</td></tr><tr><td>Microscope with objectives</td><td>Leica Microsystems CMS GmbH, Germany</td><td>DM1000, 294011-082007</td><td>histology presented in the paper was performed under ThermoFisher Scientific EVOS M5000 Imaging System, with objectives: FL 20X LWDPH, 0.40NA/3.1WD and FL 40X LWDPH 0.65NA/1.79WD</td></tr><tr><td>Myeloperoxidase from human leukocytes (MPO standard)</td><td>Merck KGaA, Darmstadt, Germany</td><td>M6908</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; x 7 H&lt;sub&gt;2&lt;/sub&gt;O (sodium phosphate dibasic heptahydrate)</td><td>Merck KGaA, Darmstadt, Germany</td><td>S9390</td><td></td></tr><tr><td>Olive-oil</td><td>Merck KGaA, Darmstadt, Germany</td><td>75343</td><td>pure, natural</td></tr><tr><td>OptEIA Mouse IFN-&amp;gamma; ELISA Set</td><td>Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA</td><td>555138</td><td></td></tr><tr><td>Ortho-dianisine dihydrochloride</td><td>Merck KGaA, Darmstadt, Germany</td><td>D3252</td><td></td></tr><tr><td>Paraffin wax</td><td>Merck KGaA, Darmstadt, Germany</td><td>76242</td><td>beads, waxy solid</td></tr><tr><td>PBS (phosphate buffered saline)</td><td>ThermoFisher Scientific,&amp;nbsp; Waltham, Massachusetts, USA</td><td>20012027</td><td>pH 7.2, mammalian cell culture</td></tr><tr><td>ph meter</td><td>Elmetron, Poland</td><td>CP-401</td><td></td></tr><tr><td>Pipettes, variable volume with tips</td><td>Merck KGaA, Darmstadt, Germany</td><td>EP3123000900-1EA</td><td>3-pack, Option 1, 0.5-10 uL/10-100 uL/100-1000 uL, includes epT.I.P.S.</td></tr><tr><td>Razor blade</td><td>VWR, Radnor, Pennsylvania, United States</td><td>PERS94-0462</td><td>scraper and cutter blades, single edge, aluminium spine, 100 blades per box, individually wrapped</td></tr><tr><td>Rotary microtome</td><td>MRC Laboratory-Instruments, Essex, CM20 2HU UK</td><td>HIS-202A</td><td></td></tr><tr><td>Scissors - straight, sharp / sharp</td><td>Animalab, Poznan, Poland</td><td>14060-10FST</td><td>Surgical instruments for procedures on mice (should be steriled)</td></tr><tr><td>Screw cap (open top)</td><td>Merck KGaA, Darmstadt, Germany</td><td>27056</td><td>black polypropylene hole cap, for use with 22&amp;nbsp;mL vial with 20-400 thread</td></tr><tr><td>Spectrophotometer</td><td>BioTek Instruments, U.S.A</td><td>201446</td><td>universal microplate spectrophotometer: &amp;lambda; range: 200 - 999 nm, absorbance measurement range: 0.000 - 4.000 Abs</td></tr><tr><td>Staining dish 20 slides with rack</td><td>Merck KGaA, Darmstadt, Germany</td><td>S6141</td><td>e.g. 20 slide staining dishes complete with covers, slide rack and handle</td></tr><tr><td>Sterile Disposable Biopsy Punch 6mm</td><td>Integra LifeSciences, Princeton, NJ, USA</td><td>33-36</td><td></td></tr><tr><td>Surgical scissors</td><td>Animalab, Poznan, Poland</td><td>52138-46</td><td>surgical instruments for procedures on mice (should be steriled)</td></tr><tr><td>Tissue processing cassettes</td><td>Merck KGaA, Darmstadt, Germany</td><td>Z672122</td><td>tissue processing/ embedding cassettes with lid</td></tr><tr><td>TMB Substrate Reagent Set</td><td>Becton Dickinson (BD Biosciences), Franklin Lakes, NJ, USA</td><td>555214</td><td></td></tr><tr><td>TNCB (2,4,6-trinitrochlorobenzene)</td><td>Tokyo Chemical Industry CO., LTD, Japan</td><td>C0307</td><td></td></tr><tr><td>TNP-BSA (bovine serum albumin conjugated with 2,4,6-trinitrophenyl)</td><td>Biosearch Technologies LGC, Petaluma, CA, USA</td><td>T-5050</td><td></td></tr><tr><td>T-PER (tissue protein extration reagent)</td><td>ThermoFisher Scientific, Waltham, Massachusetts, USA</td><td>78510</td><td></td></tr><tr><td>Tubes 15 mL sterile</td><td>Merck KGaA, Darmstadt, Germany</td><td>CLS430055 (Corning)</td><td>polypropylene, conical bottom</td></tr><tr><td>Tubes 50 mL, sterile</td><td>Merck KGaA, Darmstadt, Germany</td><td>CLS430290 (Corning)</td><td>polypropylene, conical bottom</td></tr><tr><td>Tween 20</td><td>Merck KGaA, Darmstadt, Germany</td><td>P1379</td><td></td></tr><tr><td>Vials, screw top, clear glass (vial only) 22 mL</td><td>Merck KGaA, Darmstadt, Germany</td><td>27173</td><td>for the preparation of hapten, screwed on so that it does not evaporate</td></tr><tr><td>Water bath</td><td>AJL Electronic, Poland</td><td>LW102</td><td></td></tr><tr><td>Wax (paraffin) dispenser</td><td>VWR, Radnor, Pennsylvania, United States</td><td>114-8737</td><td></td></tr><tr><td>Xylazine (xylapan 20 mg/mL) solution for injection</td><td>Vetoquinol Biowet Sp. z o.o., Gorzow Wielkopolski, Poland</td><td>cat.# not avaliable</td><td></td></tr><tr><td>Xylene (histological grade)</td><td>Merck KGaA, Darmstadt, Germany</td><td>534056</td><td></td></tr></tbody></table>

contact hypersensitivity as a murine model of allergic contact dermatitis

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an objective laboratory method that may help to study the CHS reaction in mice, which can be measured and quantified by various tests. To induce CHS, on day "0", mice were sensitized on a previously shaved spot by abdominal skin painting with the hapten 2,4,6-trinitrochlorobenzene (TNCB) in an acetone-ethanol mixture, whereas negative control mice were sham sensitized with vehicle alone-acetone-ethanol mixture. On day "4", the baseline ear thickness was measured with a micrometer prior to the elicitation of CHS (challenge) by painting both ears with diluted TNCB both in the test and control groups. After 24 h, the ear swelling was measured with a micrometer. CHS is an example of a T cell-mediated immune response that causes swelling in inflamed tissue, peaking 24 h after the skin challenge with the same hapten. An increase in ear edema correlated with augmented ear weight, myeloperoxidase (MPO) activity, pro-inflammatory cytokine concentration in the ear extracts, increased thickening of the edematous dermis in the histological examination, and ear vascular permeability. There was also an increase in the concentration of TNP-specific IgG1 antibodies in the sera of the test group when compared with the control mice. Additionally, CHS can be successfully transferred with the CHS-effector cells obtained from donors previously sensitized with TNCB. The CHS-effector cells were administered intravenously into na&#239;ve recipient mice, which were subsequently challenged with the same diluted hapten. Ear swelling was measured with a micrometer 24 h later.

Watch this Scientific Journal Video about Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis at JoVE.com

Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

Contact hypersensitivity (CHS) is an experimental model of allergic contact dermatitis (ACD) that can be studied in mice. This study aims to present an ...

contact-hypersensitivity-as-murine-model-allergic-contact

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

2.5K Views.  Wroclaw Medical University. Contact hypersensitivity (CHS) is a murine experimental model of allergic contact dermatitis (ACD). CHS is based on sensitization with reactive hapten by painting the shaved skin of the chest and abdomen, with a subsequent ear skin challenge with a diluted hapten, causing a swelling reaction that is assessed in various ways.

Video: Contact Hypersensitivity as a Murine Model of Allergic Contact Dermatitis

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical visits each year for treatment. Between 1 and 2 percent of those patients with clinical disease will experience loss of vision of the infected cornea. The vast majority of these cases are the result of reactivation of a latent infection by herpes simplex type I virus and not due to acute disease. Interestingly, the acute infection is the model most often used to study this disease. However, it was felt that a recurrent model of HSK would be more reflective of what occurs during clinical disease. The recurrent animal models for HSK have employed both rabbits and mice. The advantage of rabbits is that they experience reactivation from latency absent any known stimulus. That said, it is difficult to explore the role that many immunological factors play in recurrent HSK because the rabbit model does not have the immunological and genetic resources that the mouse has. We chose to use the mouse model for recurrent HSK because it has the advantage of there being many resources available and also we know when reactivation will occur because reactivation is induced by exposure to UV-B light. Thus far, this model has allowed those laboratories using it to define several immunological factors that are important to this disease. It has also allowed us to test both therapeutic and vaccine efficacy.

Most studies of herpetic corneal disease use a primary infection model. However, primary infection with HSV-1 does not typically lead to human disease. Here we describe a recurrent model of herpetic corneal disease, which more closely mimics human disease.

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

Analysis of Granulocyte-Macrophage Colony-Stimulating Factor-Producing T Helper Cells in a Mouse Model of Contact Hypersensitivity

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified as a distinct subset of T helper cells (GM-CSF+ IFN-&#947;- IL-17A- IL-22- effector CD4+ T cells) in human and mice. Contact hypersensitivity (CHS) is considered an excellent animal model for allergic contact dermatitis (ACD) in human, manifesting an intact T cell-mediated immune response. To provide a standardized and comprehensive assay to analyze the Th-GM cell subset in the T cell-dependent immune response in vivo, a murine CHS model was induced by sensitization/challenge with a reactive, low-molecular-weight, organic hapten, 2,4-dinitrofluorobenzene (DNFB). The Th-GM subset in effector CD4+ T cells generated upon immunization with the hapten was analyzed by flow cytometry. We found that Th-GM was mainly expanded in lesions and draining lymph nodes in the DNFB-induced CHS mouse model. This method can be applied to further study the biology of Th-GM cells and pharmacological research of therapeutic strategies centered on GM-CSF in various conditions, such as ACD.

Here, we present a simple and standardized method of analyzing the granulocyte-macrophage colony-stimulating factor-producing T helper subset in vivo.

Parallel to traditional Th1/Th2/Th17/Treg lineages, granulocyte-macrophage colony-stimulating factor-producing T helper (Th-GM) cells have been identified ...

A Mouse Ear Model for Allergic Contact Dermatitis Evaluation

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a common skin disease that manifests as a local rash, redness, and skin lesions. The occurrence and development of ACD are influenced by both genetic and environmental factors. Although many scholars have constructed a series of models of ACD in recent years, the experimental protocols of these models are all different, which makes it difficult for readers to establish them well. Therefore, a stable and efficient animal model is of great significance to further study the pathogenesis of atopic dermatitis. In this study, we detail a modeling method using 1-fluoro-2,4-dinitrobenzene (DNFB) to induce ACD-like symptoms in the ears of mice and describe several methods for assessing the severity of dermatitis during modeling. This experimental protocol has been successfully applied in some experiments and has a certain promotional role in the field of ACD research.

Here, we describe the methods for inducing allergic contact dermatitis in mouse ears by 1-fluoro-2,4-dinitrobenzene (DNFB) and how to evaluate the severity of allergic contact dermatitis.

Skin is the human body&#39;s first line of defense and one of the most exposed organs to environmental chemicals. Allergic contact dermatitis (ACD) is a ...

Citric Acid-Induced Mouse Model for Cough Hypersensitivity

Establishment of a Mouse Model with Cough Hypersensitivity via Inhalation of Citric Acid

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable economic burden. Increased cough sensitivity is a pathophysiological hallmark of chronic cough. It has been observed that cough hypersensitivity is related to airway inflammation, remodeling of airway sensory nerves, and alterations in the central nervous system. However, the precise molecular mechanisms remain unclear and require further elucidation using suitable animal models. Previous studies have utilized guinea pigs as models for studying cough, but these models present several experimental limitations, including high costs, a lack of transgenic tools, and a scarcity of commercial reagents. In addition, guinea pigs typically exhibit poor environmental tolerance and high mortality when exposed to stimuli. In contrast, mice are smaller, easier to maintain, more cost-effective, and amenable to genetic manipulation, making them more suitable for mechanistic investigations. In this study, we established a mouse model with cough hypersensitivity&#160;via&#160;continuous inhalation of citric acid (CA). This model is straightforward to operate and yields reproducible results, making it a valuable tool for further studies on the mechanisms and potential novel treatments for chronic cough.

This protocol describes the development of a mouse model with cough hypersensitivity, which can serve as an ideal model for studying the mechanisms of chronic cough.

Cough is one of the most common symptoms of many respiratory diseases. Chronic cough significantly impacts quality of life and imposes a considerable ...

Medicine

Minimum Erythematous Dose Assay: A Method for Measuring UV Sensitivity in Mouse Models

Source: Amaro-Ortiz, A. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/50670"> Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model</a>. J. Vis. Exp. (2013)
This video describes a method to measure UV sensitivity in a humanized mouse model&#8212;a mouse model that mimics human skin. We determine the minimal erythematous dose (MED) of UV radiation that can cause erythema or edema on the skin. The method can help to find out the efficacy of drugs used to reduce UV sensitivity.

Source: Amaro-Ortiz, A. et al.  Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Cancer Research

Skin Cancer

Animal model studies

A Technique to Assess the Immune Response against an Antigen by Inducing Skin Inflammation

Source: Yao, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54070">Intravital Imaging of Neutrophil Priming Using IL-1&#946; Promoter-driven DsRed Reporter Mice.</a> J. Vis. Exp. (2016).
This video demonstrates an in vivo technique to assess a test antigen by inducing skin inflammation in a murine model. The test antigen&#8212;a hapten in an oil/acetone solution&#8212;is topically applied to the skin of the mouse ear, and inflammation of the skin is assessed after an adequate period.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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