The authors would like to acknowledge Dr. Ellen M. Fox for her work regarding this model of local anaphylaxis. This work was supported by the Uniformed Services University of the Health Sciences grant number R073UE.

1. duyarlı Fareler <ol><li> 20 mg / ml &#39;lik nihai bir konsantrasyona kadar 1x PBS OVA seyreltilmesi ile bir OVA stok çözelti hazırlayın. Gelecekte kullanılmak üzere -80 ° C&#39;de cryovials ve mağaza alikotları aşağı dondurun. </li><li> Oda sıcaklığında 20 mg / mL OVA stoklar bir veya daha fazla alikotları getir. Bir kapağa sahip olan bir 5 ml polistiren yuvarlak tabanlı tüpü içinde, 1 mg / ml &#39;lik bir son konsantrasyona PBS içinde 1x OVA seyreltin. Vortex kısaca karıştırın. </li><li> : 1 oranında orta hızda ayarlanan bir girdap 1 mg / ml OVA içeren polistiren tüpü tutarken, bir 1 üretmek üzere tüp vorteks devam ederken, iyice homojenleştirilmiştir Imject Alum (40 mg / ml) damla damla, bir eşit hacim kazandırmak immünojene Şap. </li><li> Girdaba boru ve boru üzerinde bant kapağını yerleştirin. Düşük ayarda vorteks açın ve OVA 30 dakika boyunca Alum&#39;un tutunmasını sağlar. <ol><li> Zaman büyük miktarda OVA / Alum hazırlanması ve fareler hassaslaştıncı arasında geçmesine izin vermeyin. Hassaslaşması OVA / Şap preparatın sonra doğrudan tamamlanmış edilemezseOda sıcaklığında iyonu deposu OVA / şap. Kullanmadan önce, 1 dakika vorteksleyin. </li></ol></li><li> OVA / Şap karışımı ile 1 ml insülin şırınga yükleyin. 27 gauge&#39;lik bir iğne kullanılarak şırınga kapağı. </li><li> OVA &#39;nın 50 ug Alum ve 2 mg fare başına bir doz için, BALB / c farelerinin periton içine OVA / Alum 100 ul enjekte edilir. </li><li> Negatif kontrol için, PBS uygulanan farelerin başka bir grup duyarlı. OVA çıkararak Yukarıda listelendiği gibi aynı şekilde Alum ve PBS&#39;nin 1: 1 karışımı hazırlayın. </li><li> 3 enjeksiyondan toplam 3 hafta boyunca haftada bir kez tüm fareleri (OVA ve PBS kontrol) duyarlı. Son enjeksiyondan sonra, yerel anafilaksi deneyini gerçekleştirmeden önce en az 2 hafta bekleyin. </li></ol> 2. Yerel Anafilaksi Deneyi <ol><li> Oda sıcaklığına OVA stok çözüm getirecek. (: 1X PBS OVA 4 seyreltme 1), 1.5 ml mikrosantrifüj tüpü olarak, PBS ile 5 mg / ml &#39;lik nihai bir konsantrasyona kadar 20 mg / ml stok OVA seyreltin. Vortex kısaca karıştırın. </li><li> RC 2 &lt;kullanarak fareler anestezi/ Sup&gt; Kemirgen Anestezi Sistemi (veya benzer buharlaşmış dağıtım sistemi). Izofluran ile doldurun, ve O 2 hava akımını açın. Indüksiyon odasında Yeri farenin ve Anesthetization başlatmak için% 5 izofluran kumanda düğmesini ayarlayın. Farenin tamamen uyuşturulduktan sonra, hareketlilik eksikliği ile gösterilen, derin Anesthetization korumak için% 3 izofluran kumanda düğmesini ayarlayın. Bu prosedürde anestezi amacı, hayvan hareketsizleştirmektir. Kulak ve kuyruk ven enjeksiyonları hayvana ciddi ağrı veya sıkıntı yok. NOT: İsofluranın insan üreme sistemi üzerinde olumsuz etkileri olabilir. Atık gazın etkisiz hale getirmek için iyi havalandırılan bir odada kullanmak ve indüksiyon odasına bir atma bidonu eklemek için emin olun. Izofluran kullanırken hamile iseniz, maruziyeti en aza indirmek için bir karbon filtre maske takmalıdır. </li><li> Yük bir 31 G iğne ile kapatılmış bir 3/10 cc&#39;lik bir şırıngaya insülin 5 mg / ml OVA 10 ul. 10 ul başka bir 3/10 cc şırınga insülin yük1x PBS ile yıkandı. </li><li> Diseksiyon mikroskobu kullanarak, OVA 50 ug bir meydan okuma doz için bir anestezi fare sağ kulağına OVA 10 ul enjekte. Diğer dozlar, örneğin, 20 ug olarak da kabul edilebilir. <ol><li> 15 ml konik tüp etrafında seloteyp bant (yapışkan tarafı yukarı) wrap, enjeksiyon için kulak stabilize etmek. Teybe sağ kulağının dorsal yüzü sabitleyin. Kulak, iki yaprak arasındaki iğne konik tarafı yerleştirin. Yavaşça herhangi bir kan damarlarını vurmak için özen, kulak doku merkezi haline OVA enjekte. </li></ol></li><li> Yukarıda tarif edilen aynı yöntem kullanılarak, bir negatif kontrol olarak, sol kulağa 10 ul PBS enjekte edilir. </li><li> 3 dakika bekleyin. <ol><li> Bu süre içinde, bir süzgeç içinde fare ve 1 dakika kan damarlarını genişletmek için bir ısı lambası altına veya sıcak su banyosu içinde kuyruk tutun. </li></ol></li><li> Yavaşça kuyruk v içine% 0.5 Evans mavisi 200 ul enjekte bir 27 G iğne ile kapatıldı 1 ml&#39;lik şırınga kullanarak insülinfare ein. Tüm 200 ul önce damar yapısını tahrip olabilir Rapid enjeksiyonu uygulanır. Bu durumda, kuyruk karşı tarafında ven içine boyanın geri kalan hacim enjekte çalışır. </li><li> 10 dakika bekleyin. </li><li> Fareyi Euthanize ve forseps ve cerrahi makas kullanılarak hem kulaklarını çıkarın. <ol><li> Basınç kulak merkezinde enjeksiyon sahaya uygulanır; emin, forseps ile kulak kenarları al. Yakın kesim ile mümkün olduğu kadar çok kulak doku çıkarın, ancak boydan boya olmayabilen, kulak tabanında kıkırdak mevcut olan sırt. Kürk küçük miktarlarda kesilerek çıkarılmış kulak üzerinde mevcut olabilir; Bu, deneyin etkilemez. </li></ol></li><li> Kısım bir 1.5 ml mikrosantrifüj tüpleri içine formamit 700 ul. Kulak başına bir tüp (fare başına iki tüp) hazırlayın. NOT: Formamide, teratojenik, mutajenik ve tahriş edicidir. Eldiven, koruyucu gözlük ve yüz koruyucu giyin. Bir tutarken Hamile kadınlar dikkatli kullanmalıdırs formamıd fetotoksik olabilir. </li><li> , Formamıd tüpler kap ve 63 ° C&#39;ye ayarlanmış kuru bir ısı banyosu gecede inkübe kulakları ekleyin. Kulaklar hala kuluçkadan sonra biraz mavi görünebilir; Ancak boyanın çoğunluğu formamid içine ekstre edilir. </li><li> 96 oyuklu bir plaka için, iki kopya halinde, her bir numune 300 ul ilave edin. Bu numune içerisinde mevcut olması halinde kürk aktarılmasını önlemek. Kuyu içinde mevcut hava kabarcığı vardır ki bunlar absorbans okumasını etkileyecek şekilde sağlanması. </li><li> 96 gözlü plaka, iki kopya halinde, formamid 300 ul ilave edin. Bu kuyular, blok olarak kullanılacaktır. </li><li> 620 nm&#39;de absorbans ölçümü için bir spektrofotometre kullanılır. Her örnek okuma Çıkar formamiddir kuyular. Not: Bütün deneyler Kuvvetler Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi tarafından onaylanmış protokolleri altında yapıldı. </li></ol>

<ol>
	<li>Kojima, T., 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=Mast+cells+and+Bbasophils+are+selectively+activated+in+vitro+and+in+vivo+through+CD200R3+in+an+IgE-independent+manner.">Mast cells and Bbasophils are selectively activated in vitro and in vivo through CD200R3 in an IgE-independent manner.</a> The Journal of Immunology. 179 (10), 7093-7100 (2007).</li><li>Parikh, S. A., Cho, S. H., Oh, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preformed+enzymes+in+mast+cell+granules+and+their+potential+role+in+allergic+rhinitis.">Preformed enzymes in mast cell granules and their potential role in allergic rhinitis.</a> Current Allergy and Asthma Reports. 3 (3), 266-272 (2003).</li><li>Amin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mast+cells+in+allergic+inflammation.">The role of mast cells in allergic inflammation.</a> Respiratory Medicine. 106 (1), 9-14 (2012).</li><li>Nakaya, M., Takeuchi, N., Kondo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+localization+of+histamine+receptor+subtypes+in+human+inferior+turbinates.">Immunohistochemical localization of histamine receptor subtypes in human inferior turbinates.</a> Annals of Otology, Rhinology &amp; Laryngology. 113 (7), 552-557 (2004).</li><li>Ramsdell, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+Trypan+Blue+to+demonstrate+the+immediate+skin+reaction+in+rabbits+and+guinea+pigs.">The use of Trypan Blue to demonstrate the immediate skin reaction in rabbits and guinea pigs.</a> The Journal of Immunology. 15 (4), 305-311 (1928).</li><li>Chase, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+sensitization+of+animals+with+simple+chemical+compounds:+X.+Antibodies+inducing+immediate-type+skin+reactions.">Studies on the sensitization of animals with simple chemical compounds: X. Antibodies inducing immediate-type skin reactions.</a> The Journal of Experimental Medicine. 86 (6), 489-514 (1947).</li><li>Goose, J., Blair, A. M. J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+rat,+induced+with+two+homologous+reagin-like+antibodies+and+its+specific+inhibition+with+disodium+cromoglycate.">Passive cutaneous anaphylaxis in the rat, induced with two homologous reagin-like antibodies and its specific inhibition with disodium cromoglycate.</a> Immunology. 16, 749-760 (1969).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passive+cutaneous+anaphylaxis+in+the+mouse.">Passive cutaneous anaphylaxis in the mouse.</a> The Journal of Immunology. 81 (4), 355-357 (1958).</li><li>Ovary, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immediate+reactions+in+the+skin+of+experimental+animals+provoked+by+antibody-antigen+interactoin.">Immediate reactions in the skin of experimental animals provoked by antibody-antigen interactoin.</a> Progress in Allergy. 5, 459-508 (1958).</li><li>Akiyama, 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=Quantitiative+evaluation+of+passive+cutaneous+anaphylaxis+(PCA)+using+a+hand-held+spectrophotometer.">Quantitiative evaluation of passive cutaneous anaphylaxis (PCA) using a hand-held spectrophotometer.</a> Biological &amp; Pharmaceutical Bulletin. 19 (8), 1112-1114 (1996).</li><li>Teshima, R., 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=Simple+spectrophotometric+analysis+of+passive+and+active+ear+cutaneous+anaphylaxis+in+the+mouse.">Simple spectrophotometric analysis of passive and active ear cutaneous anaphylaxis in the mouse.</a> Toxicology Letters. 95 (2), 109-115 (1998).</li></ol>

The authors have nothing to disclose.

Alerjik hastalıkların sayıda belirteçleri havayolu meydan ortamda bronkoalveolar sıvı dolaşması histamin Th2 sitokin üretimi ve hücre işe seviyelerindeki değişiklikler dahil olmak üzere, alerji yapıcının aşağıdaki alerjik cevaplar, gücünü değerlendirmek için kullanılır. Bu parametrelerdeki değişiklikleri izleme alerjik reaksiyonlara eğitim için önemli olmakla birlikte, biyolojik belirteçlerin her zaman klinik alerjik hastalığı ile ilişkili değildir. Bu protokol açıklanan yerel ana...

Tip I hipersensitivite, mast hücrelerinin ve bazofillerin yüzeyi üzerindeki IgE, antijen ile indüklenen çapraz-bağlanma ile aracılık eder. Bu hücre degranülasyonunu ve histamin, triptaz ve trombosit aktive edici faktör gibi vazoaktif 2 ve proenflamatuvar medyatörlerin salınmasına yolaçar. Degranülasyonunun sırasında önceden oluşturulmuş aracıların yayımlanmasının ardından, mast hücreleri sentez ve daha fazla damar geçirgenliği 3 artış sağlayan prostaglandin ve lökotrienler bırakın. İlk klinik yanıt hızlı bir şekilde oluşur ve &quot;acil reaksiyonu&quot; olarak adlandırılır. Deride, bir pervaz ve şişlik tepki antijenin dakika içinde kolayca görülebilir olduğu anlamındadır. Meydan dozuna bağlı olarak, bir &quot;geç faz tepkisi&quot; bir kaç saat sonra görmek mümkündür. Geç faz şişlik dokulara 2 içine lokalize ödem ve lökosit işe kaynaklanmaktadır. Histamin, genellikle katılan önemli arabulucu olarak kabulani alerjik tepkiler, 1 (HR1) kaplar ve histamin reseptörü 2 (HR2) düz kasında eksprese ifade histamin reseptörü üzerine etki eder. Kombine etki, inflamasyon 4 bölgesinde kan akışını ve damar geçirgenliğini artırır. Alerji hayvan modellerinde çeşitli alerjik astım, sistemik anafilaksi ve yerel anafilaksi modelleri dahil alerjik iltihabı dahil mekanizmaları, incelemek amacıyla geliştirilmiştir. Intravenöz boya uygulama yayınlar 1920&#39;lerde 5 geri Bu teknik Bayan açıklayan, neredeyse bir yüzyıl için hayvan modellerinde lokalize alerjik tepkileri ölçmek için kullanılır olmuştur. Tavşanlar ve kobay ani hipersensitivite reaksiyonları test etmek için kullanılan ilk hayvan modelleri vardı ve en hassas yanıtlar genellikle kulakta 5,6 bulundu. Tahlil daha sonra sıçanlarda 7 ve farelerde 8 kullanılmak üzere onaylandı. Tarihsel olarak,Deney çeşitli yöntemler önceki boya enjeksiyon, boya enjeksiyondan önce antijen enjeksiyonundan antijen enjeksiyonu ve boya ve antijenin eş zamanlı olarak enjeksiyonu da dahil olmak üzere, kullanılmıştır. Alerjik yanıtların ölçülmesi için bir vasıta olarak intravenöz uygulama boya pasif ve aktif ölçmek için ikinci ve pasif reaksiyonlar 5,9 ters edilebilir çok yönlü bir deneyidir. Sayısız boyalar Tripan Blue, Pontamine Sky Blue, Evans Blue, Geigy Mavi 536 ve Hindistan Mürekkep 5,6,9 dahil alerjik yanıtları, değerlendirmek için kullanılmıştır. % 0.5 Evans mavisi çözeltisi halen deri alerjik yanıtı ölçmek için kullanılan standart bir boyadır. Meydan anafilaktik cevap kısa süreli olduğu; maksimum yoğunluğu, 10 içinde ulaşılır - boya enjeksiyonunun 15 dakika ve boya ne olursa olsun, kullanılan 9 hayvan türlerinin, cebine fazla 30 dakika tatbik edilirse, herhangi bir tepki görülebilir. Boya ekstravazasyon Kantitasyonu özgünıy mavi boya 7-9 ile gösterildiği gibi kabarıklık büyüklüğü ölçülerek elde edilmiştir. Buna ek olarak, granüle mast hücre sayımları, reaksiyon ortamının cilt dokusunun kesilmesi ve 7 mavi toluidin ile lekeleme ile tayin edilebilir. Mast hücreleri, yüksek afinite IgE reseptörü eksprese FcεRI ana yerel hücre popülasyonu olarak mast hücre degranülasyonu, genellikle, deri altı, IgE aracılı alerjik karşılıkları için bir işaretleyici olarak kullanılmaktadır. Doku içine boya ekstravazasyonunu ölçmek için spektrofotometrik teknikler 1990 yılında sıçan 10 ve fare 11 pasif deri anafilaksi (PCA) için geliştirilmiştir. Aşağıda yerel anafilaksi Deney protokolü. Kojima ve bunlar 1 ila uyarlanmış ve allerjik tepkileri ortaya çıkarmak için antijen olarak tavuk yumurtası (OVA) kullanır edildi. Bununla birlikte, istenirse OVA başka antijenler de kullanılabilir. Tahlil sonra vasküler Geçirgenlik değişiklikleri izlemek için Evans Mavi boya kullanırHücre IgE çapraz bağlanması ve histamin salgısını mast nedeniyle meydana y.

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Name of Material/ Equipment</td>
			<td style="width:167px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:225px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">BALB/c Mice</td>
			<td style="width:167px;">The Jackson Laboratory</td>
			<td style="width:135px;">651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PBS pH 7.4</td>
			<td style="width:167px;">Quality Biologic</td>
			<td style="width:135px;">114-058-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ovalbumin</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">A5503-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Imject Alum</td>
			<td style="width:167px;">Thermo Scientific</td>
			<td style="width:135px;">77161</td>
			<td style="width:225px;">Mix thoroughly before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Evans Blue Dye</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">E2129</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Formamide</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:135px;">295876</td>
			<td style="width:225px;">99%+ Spectrophotometric grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Isoflurane, USP</td>
			<td style="width:167px;">Phoenix</td>
			<td style="width:135px;">NDC 57319-474-06</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1cc Insulin syringes</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">329654</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">3/10 cc Insulin syringe with 31G needle</td>
			<td style="width:167px;">Terumo</td>
			<td style="width:135px;">NDC 100861</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27G Needles</td>
			<td style="width:167px;">BD</td>
			<td style="width:135px;">305109</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Forceps</td>
			<td style="width:167px;">F.S.T.</td>
			<td style="width:135px;">11000-12</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Surgical scissors</td>
			<td style="width:167px;">F.S.T.</td>
			<td style="width:135px;">14070-12</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">5ml Polystyrene round-bottom tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352058</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1.5ml Microcentrifuge tubes</td>
			<td style="width:167px;">Medical Supply Partners</td>
			<td style="width:135px;">15-1151</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">15ml Conical tubes</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:135px;">352097</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Flat-bottom 96 well plate</td>
			<td style="width:167px;">Costar</td>
			<td style="width:135px;">3590</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Scotch tape</td>
			<td style="width:167px;"></td>
			<td style="width:135px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:271px;">RC2 Rodent Anesthesia System</td>
			<td style="width:167px;">VetEquip</td>
			<td style="width:135px;">922100</td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Vortex Genie 2</td>
			<td style="width:167px;">Scientific Industries</td>
			<td style="width:135px;">SI-0236</td>
			<td style="width:225px;">Model G560 with 3 inch platform</td>
		</tr>
	</tbody>
</table>

measuring local anaphylaxis in mice

Alerjik tepkiler, mast hücreleri ve bazofiller ve vazoaktif ve proenflamatuvar medyatörlerin, bunu takiben de aktivasyonu sonucudur. Bir hassaslaşmış bireyde bir alerjene maruz kalma küçük eritemden hayatı tehdit eden anafilaksi farklılık klinik semptomlara neden olabilirler. Laboratuvarda, çeşitli hayvan modelleri alerjik yanıtları sürüş mekanizmalarını anlamak için geliştirilmiştir. Bu yazıda, lokalize alerjik yanıtları ölçmek için damar geçirgenliği değişiklikleri ölçmek için ayrıntılı bir yöntem açıklanmaktadır. Yerel anafilaksi tahlil, ilk 1920&#39;lerde bildirildi ve Kojima ve arkadaşları tarafından yayınlanan tekniği adapte edilmiştir. 2007 yılında 1. Bu testte, OVA duyarlı farenin araç ile sol kulak ve OVA ile sağ kulakta zorlanmaktadır. Bu Evans Mavisi damardan enjeksiyonu ile takip edilir. On dakika sonra enjekte Evans mavisi, hayvanda damar dışına olan boya ötenazi yapılmış ve birkulaklara formamidde gecede ekstre edilir. Ekstre boyanın absorbans daha sonra bir spektrofotometre ile ölçülür. Bu yöntem güvenilir bir lokal allerjik yanıtın görsel ve ölçülebilir tezahürü sonuçlanır.

Başarıyla tahlil geçirmiş hayvanların deri ve mavi görünür gözleri vardır. PBS duyarlı hayvanlar bu nedenle hem kulaklar beyaz (Şekil 1A) kalmalıdır, PBS veya OVA da tepki olmamalıdır. OVA hassas hayvanlarda PBS meydan alan kulak (sol) tamamen beyaz veya enjeksiyon bölgesinde lokal bir şekilde hafifçe mavi olarak olmalıdır. OVA alan kulak (sağ) bir boya uygulama için (Şekil 1B) sonra 10 dakika boyunca aşamalı olarak koyu mavi olmalıdır. <p class="jove_con...

Watch this Scientific Journal Video about Measuring Local Anaphylaxis in Mice at JoVE.com

Measuring Local Anaphylaxis in Mice

Mast hücreleri ve bazofillerin aktivasyonuyla karakterize alerjik tepkiler, IgE çapraz bağlanması ve proenflamatuar aracıların salınması ile tahrik edilmektedir. Alerjik tepkilerin bir kantitatif olarak değerlendirilmesi allerjen vasküler geçirgenlikte değişiklikleri izlemek için Evans mavisinin kullanılması sureti ile elde edilebilir.

Farelerde Yerel Anafilaksi Ölçme

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

Research

JoVE Journal

Immunology and Infection

19.1K Görüntüleme.  Uniformed Services University of the Health Sciences.  Mast hücreleri ve bazofillerin aktivasyonuyla karakterize alerjik tepkiler, IgE çapraz bağlanması ve proenflamatuar aracıların salınması ile tahrik edilmektedir. Alerjik tepkilerin bir kantitatif olarak değerlendirilmesi allerjen vasküler geçirgenlikte değişiklikleri izlemek için Evans mavisinin kullanılması sureti ile elde edilebilir. 

Video: Measuring Local Anaphylaxis in Mice

Measuring Calpain Activity in Fixed and Living Cells by Flow Cytometry

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, including signaling, cytoskeletal remodeling, regulation of gene expression, apoptosis and cell cycle progression 1. Calpains have been implicated in many pathologies including muscular dystrophies, cancer, diabetes, Alzheimer's disease and multiple sclerosis 1. Calpain regulation is complex and incompletely understood. mRNA and protein levels correlate poorly with activity, limiting the use of gene or protein expression techniques to measure calpain activity. This video protocol details a flow cytometric assay developed in our laboratory for measuring calpain activity in fixed and living cells. This method uses the fluorescent substrate BOC-LM-CMAC, which is cleaved specifically by calpain, to measure calpain activity. 2 In this video, calpain activity in fixed and living murine 32Dkit leukemia cells, alone or as part of a splenocyte population is measured using an LSRII (BD Bioscience). 32Dkit cells are shown to have elevated activity compared to normal splenocytes.

This article will detail the protocol for measuring calpain activity in fixed and living cells using flow cytometry.

Sabit ve Yaşam Hücreler Sitometrisi ile Calpain Aktivitesi Ölçme

Calpains are ubiquitous intracellular, calcium-sensitive, neutral cysteine proteases 1. Calpains play crucial roles in many physiological processes, ...

İmmünoloji ve Enfeksiyon

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of Listeria, which results in systemic infection2. After injection, Listeria quickly disseminates to the spleen and liver due to uptake by CD8&alpha;+ dendritic cells and Kupffer cells3,4. Once phagocytosed, various bacterial proteins enable Listeria to escape the phagosome, survive within the cytosol, and infect neighboring cells5. During the first three days of infection, different innate immune cells (e.g. monocytes, neutrophils, NK cells, dendritic cells) mediate bactericidal mechanisms that minimize Listeria proliferation. CD8+ T cells are subsequently recruited and responsible for the eventual clearance of Listeria from the host, typically within 10 days of infection6.
Successful clearance of Listeria from infected mice depends on the appropriate onset of host immune responses6	. There is a broad range of sensitivities amongst inbred mouse strains7,8. Generally, mice with increased susceptibility to Listeria infection are less able to control bacterial proliferation, demonstrating increased bacterial load and/or delayed clearance compared to resistant mice. Genetic studies, including linkage analyses and knockout mouse strains, have identified various genes for which sequence variation affects host responses to Listeria infection6,8-14. Determination and comparison of infection kinetics between different mouse strains is therefore an important method for identifying host genetic factors that contribute to immune responses against Listeria. Comparison of host responses to different Listeria strains is also an effective way to identify bacterial virulence factors that may serve as potential targets for antibiotic therapy or vaccine design.
We describe here a straightforward method for measuring bacterial load (colony forming units [CFU] per tissue) and preparing single-cell suspensions of the liver and spleen for FACS analysis of immune responses in Listeria-infected mice. This method is particularly useful for initial characterization of Listeria infection in novel mouse strains, as well as comparison of immune responses between different mouse strains infected with Listeria. We use the Listeria monocytogenes EGD strain15 that, when cultured on blood agar, exhibits a characteristic halo zone around each colony due to &beta;-hemolysis1 (Figure 1). Bacterial load and immune responses can be determined at any time-point after infection by culturing tissue homogenate on blood agar plates and preparing tissue cell suspensions for FACS analysis using the protocols described below. We would note that individuals who are immunocompromised or pregnant should not handle Listeria, and the relevant institutional biosafety committee and animal facility management should be consulted before work commences.

Measuring Bacterial Load and Immune Responses in Mice Infected with Listeria monocytogenes

Listeria monocytogenes is a model organism for studying immune responses and genetic susceptibility to intracellular bacteria in mice. This method enables one to measure bacterial load and generate single-cell suspensions of the liver and spleen from mice for FACS analysis to determine changes in immune cells due to Listeria infection.

Ile Enfekte Fare Bakteriyel Yük ve Bağışıklık Yanıtları Ölçüm Listeria monocytogenes</em

Listeria monocytogenes (Listeria) is a Gram-positive facultative intracellular pathogen1. Mouse studies typically employ intravenous injection of ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

Measurement of &amp;#947;HV68 Infection in Mice

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

Farelerde γHV68 Enfeksiyon ölçümü

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

İnsan Kanı Ölçüm Granülosit ve Monosit Fagositoz ve Oksidatif Burst Etkinlik

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Measuring Deformability and Red Cell Heterogeneity in Blood by Ektacytometry

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of disease or occurrence of serious complications. Ektacytometry uses laser diffraction viscometry to measure the deformability of red blood cells subject to either increasing shear stress or an osmotic gradient at a constant value of applied shear stress. However, direct deformability measurements are difficult to interpret when measuring heterogenous blood that is characterized by the presence of both rigid and deformable red cells. This is due to the inability of rigid cells to properly align in response to shear stress and results in a distorted diffraction pattern marked by an exaggerated decrease in apparent deformability. Measurement of the degree of distortion provides an indicator of the heterogeneity of the erythrocytes in blood. In sickle cell anemia, this is correlated with the percentage of rigid cells, which reflects the hemoglobin concentration and hemoglobin composition of the erythrocytes. In addition to measuring deformability, osmotic gradient ektacytometry provides information about the osmotic fragility and hydration status of erythrocytes. These parameters also reflect the hemoglobin composition of red blood cells from sickle cell patients. Ektacytometry measures deformability in populations of red cells and does not, therefore, provide information on the deformability or mechanical properties of individual erythrocytes. Regardless, the goal of the techniques described herein is to provide a convenient and reliable method for measuring the deformability and cellular heterogeneity of blood. These techniques may be useful for monitoring temporal changes, as well as disease progression and response to therapeutic intervention in several disorders. Sickle cell anemia is one well-characterized example. Other potential disorders where measurements of red cell deformability and/or heterogeneity are of interest include blood storage, diabetes, Plasmodium infection, iron deficiency, and the hemolytic anemias due to membrane defects.

Here we present techniques to measure red cell deformability and cellular heterogeneity by ektacytometry. These techniques are applicable to general investigations of red cell deformability and specific investigations of blood diseases characterized by the presence of both rigid and deformable red cells in circulation, such as sickle cell anemia.

Deformasyon ve kan Ektacytometry tarafından kırmızı hücre heterojenite ölçme

Decreased red cell deformability is characteristic of several disorders. In some cases, the extent of defective deformability can predict severity of ...

Characterization of Thymus-dependent and Thymus-independent Immunoglobulin Isotype Responses in Mice Using Enzyme-linked Immunosorbent Assay

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a formidable defense against invading pathogens via diverse mechanisms. One major goal of vaccination is to induce protective antigen-specific antibodies to prevent life-threatening infections. Both thymus-dependent (TD) and thymus-independent (TI) antigens can elicit robust antigen-specific IgM responses and can also induce the production of isotype-switched antibodies (IgG, IgA and IgE) as well as the generation of memory B cells with the help provided by antigen presenting cells (APCs). Here, we describe a protocol to characterize TD and TI Ig isotype responses in mice using enzyme-linked immunosorbent assay (ELISA). In this protocol, TD and TI Ig responses are elicited in mice by intraperitoneal (i.p.) immunization with hapten-conjugated model antigens TNP-KLH (in alum) and TNP-polysaccharide (in PBS), respectively. To induce TD memory response, a booster immunization of TNP-KLH in alum is given at 3 weeks after the first immunization with the same antigen/adjuvant. Mouse sera are harvested at different time points before and after immunization. Total serum Ig levels and TNP-specific antibodies are subsequently quantified using Ig isotype-specific Sandwich and indirect ELISA, respectively. In order to correctly quantify the serum concentration of each Ig isotype, the samples need to be appropriately diluted to fit within the linear range of the standard curves. Using this protocol, we have consistently obtained reliable results with high specificity and sensitivity. When used in combination with other complementary methods such as flow cytometry, in vitro culture of splenic B cells and immunohistochemical staining (IHC), this protocol will allow researchers to gain a comprehensive understanding of antibody responses in a given experimental setting.

In this paper, we describe a protocol to characterize T-dependent and T-independent immunoglobulin (Ig) isotype responses in mice using ELISA. This method used alone or in combination with flow cytometry will allow researchers to identify differences in B cell-mediated Ig isotype responses in mice following T-dependent and T-independent antigen immunization.

Timus bağlı ve timus bağımsız immünglobulin izotip yanıt Immunosorbent Assay enzim bağlı kullanarak farelerde karakterizasyonu

Antibodies, also termed as immunoglobulins (Ig), secreted by differentiated B lymphocytes, plasmablasts/plasma cells, in humoral immunity provide a ...

Mouse Body Temperature Measurement Using Infrared Thermometer During Passive Systemic Anaphylaxis and Food Allergy Evaluation

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature readings is common, and they have been proven to be accurate and invaluable in this regard. However, this method of temperature measurement requires the mice to be anesthetized in order to insert the probe without injury to the animal. This limits the ability to observe other phenotypes of the mouse simultaneously. In order to investigate other phenotypes while measuring temperatures, rectal probes are not ideal, and another method is desired. Here, we introduce a noninvasive method of temperature measurement that foregoes the requirement for mouse anesthesia while maintaining equal reliability to rectal probes in measuring body temperature. We use an infrared thermometer that detects body surface temperatures at ranges between 2 and 150 mm. This method of body temperature measurement is successful in reliably replicating temperature change trends during passive system anaphylaxis experiments in mice. We show that body surface temperatures are about 2.0 &#176;C lower than rectal probe measurements, but the degree of temperature drop follows the same trend. Furthermore, we use the same technique to observe mice in a food allergy model to evaluate temperature and activity levels simultaneously.

Here we present a new method to accurately measure body temperature differences in passive systemic anaphylaxis (PSA) and food allergy mouse models using an infrared thermometer. This procedure has been accurately duplicated in previous PSA results.

Kızılötesi termometre pasif sistemik anafilaksi ve gıda Anti değerlendirme sırasında kullanarak fare vücut sıcaklık ölçümü

Mouse body temperature measurement is of paramount importance for investigating allergies and anaphylactic symptoms. Rectal probes for temperature ...

Quantification of Atherosclerosis in Mice

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic inflammatory disease. Experimental atherosclerosis studies have elucidated the role of cholesterol and inflammation in the disease process. This has led to successful clinical trials with pharmaceutical agents that reduce clinical manifestations of atherosclerosis. Careful and well-controlled experiments in mouse models of the disease could further elucidate the pathogenesis of the disease, which is not fully understood. Standardized lesion analysis is important to reduce experimental variability and increase reproducibility. Determining lesion size in aortic root, aortic arch, and brachiocephalic artery are common endpoints in experimental atherosclerosis. This protocol provides a technical description for evaluation of atherosclerosis at all these sites in a single mouse. The protocol is particularly useful when material is limited, as is frequently the case when genetically modified animals are being characterized.

Murine models of atherosclerosis are useful tools to investigate pathogenic pathways on a molecular level, but require standardized quantification of lesion development. This protocol describes an optimized method to determine lesion size in the major arterial vessels including the aortic root, aortic arch, and brachiocephalic artery.

Fareler içinde ateroskleroz ölçümü

Cardiovascular disease is the main cause of death in the world. The underlying cause in most cases is atherosclerosis, which is in part a chronic ...

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase (G6PI)-Induced RA Mice

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T (iNKT) cells. Patients with active RA present fewer iNKT cells, defective cell function, and excessive polarization of Th1. In this study, an RA animal model was established using a mixture of hGPI325-339 and hGPI469-483 peptides. The iNKT cells were obtained by in vivo induction and in vitro purification, followed by infusion into RA mice for adoptive immunotherapy. The in vivo imaging system (IVIS) tracking revealed that iNKT cells were mainly distributed in the spleen and liver. On day 12 after cell therapy, the disease progression slowed down significantly, the clinical symptoms were alleviated, the abundance of iNKT cells in the thymus increased, the proportion of iNKT1 in the thymus decreased, and the levels of TNF-&#945;, IFN-&#947;, and IL-6 in the serum decreased. Adoptive immunotherapy of iNKT cells restored the balance of immune cells and corrected the excessive inflammation of the body.

Adoptive Immunotherapy of iNKT Cells in Glucose-6-Phosphate Isomerase G6PI-Induced RA Mice

This protocol uses G6PI mixed peptides to construct rheumatoid arthritis models that are closer to that of human rheumatoid arthritis in CD4+ T cells and cytokines. High purity invariant natural killer T cells (mainly iNKT2) with specific phenotypes and functions were obtained by in vivo induction and in vitro purification for adoptive immunotherapy.

Glukoz-6-Fosfat İzomazlarında (G6PI)-Indüklenen RA Farelerde iNKT Hücrelerinin Benimseyici İmmünoterapisi

Rheumatoid arthritis (RA) is a complex chronic inflammatory autoimmune disease. The pathogenesis of the disease is related to invariant natural killer T ...

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are cleared from the human lung alveoli by being phagocytosed by innate monocytes and/or neutrophils. This protocol offers a fast and reliable measurement of phagocytosis by flow cytometry using fluorescein isothiocyanate (FITC)-labeled conidia for co-incubation with human leukocytes and subsequent counterstaining with an anti-FITC antibody to allow discrimination of internalized and cell-adherent conidia. Major advantages of this protocol are its rapidness, the possibility to combine the assay with cytometric analysis of other cell markers of interest, the simultaneous analysis of monocytes and neutrophils from a single sample and its applicability to other cell wall-bearing fungi or bacteria. Determination of percentages of phagocytosing leukocytes provides a means to microbiologists for evaluating virulence of a pathogen or for comparing pathogen wildtypes and mutants as well as to immunologists for investigating human leukocyte capabilities to combat pathogens.

Measuring Phagocytosis of Aspergillus fumigatus Conidia by Human Leukocytes using Flow Cytometry

This protocol provides a fast and reliable method to quantitatively measure phagocytosis of Aspergillus fumigatus conidia by human primary phagocytes using flow cytometry and to discriminate phagocytosis of conidia from mere adhesion to leukocytes.

Akış Sitometrisi Kullanılarak İnsan Lökositler tarafından Aspergillus fumigatus Conidia Fagositozunun ölçülmesi

Invasive pulmonary infection by the mold Aspergillus fumigatus poses a great threat to immunocompromised patients. Inhaled fungal conidia (spores) are ...