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. Sensibilizar Ratos <ol><li> Prepara-se uma solução estoque de OVA por diluição OVA em 1x PBS para uma concentração final de 20 mg / ml. Congelar as alíquotas em criotubos e armazenar a -80 ° C para uso futuro. </li><li> Trazer uma ou mais alíquotas de 20 mg / ml de caldo de OVA à temperatura ambiente. Dilui-se com OVA em 1x PBS para uma concentração final de 1 mg / ml num tubo de 5 ml de fundo redondo de poliestireno com uma tampa. Vortex para misturar brevemente. </li><li> Enquanto mantém o tubo de poliestireno contendo a 1 mg / ml de OVA sobre um vórtice à velocidade média, adicionar um volume igual de completamente homogeneizada Imject Alum (40 mg / ml), gota a gota, enquanto continua a vortex do tubo para produzir uma proporção de 1: 1 de Alum de imunogénio. </li><li> Coloque a tampa no tubo e fita de tubo de vórtice. Vire vórtice em configuração mais baixa e permitir OVA de adsorção ao Alum por 30 min. <ol><li> Não permita que uma grande quantidade de tempo para passar entre a preparação OVA / Alum e sensibilizar camundongos. Se sensibilização não pode ser preenchido diretamente após OVA / Alum preparation, loja OVA / Alum à temperatura ambiente. Vortex por 1 min antes de usar. </li></ol></li><li> Coloque um 1 ml seringa de insulina com a mistura de OVA / Alum. Tapar a seringa com agulha de calibre 27. </li><li> Injectar 100 uL de OVA / alum no peritoneu de ratinhos BALB / c por uma dose por ratinho de 50 ug de OVA e 2 mg de alúmen. </li><li> Para um controlo negativo, sensibilizar outro grupo de ratinhos a PBS. Prepara-se uma mistura 1: 1 de Alum e PBS no mesmo modo como listado acima, omitindo OVA. </li><li> Sensibilizar todos os ratos (controle OVA e PBS) uma vez por semana, durante três semanas para um total de 3 injeções. Após a injeção final, esperar pelo menos duas semanas antes da realização do ensaio anafilaxia local. </li></ol> 2. local Anafilaxia Assay <ol><li> Levar a solução estoque de OVA à temperatura ambiente. Num tubo de microcentrífuga de 1,5 ml, diluir a 20 mg / ml de caldo de OVA a uma concentração final de 5 mg / ml com PBS (diluição 1: 4 de OVA em PBS 1X). Vortex para misturar brevemente. </li><li> Anestesiar ratos usando RC 2 &lt;/ Sup&gt; Rodent Sistema de Anestesia (ou sistema de entrega vaporizado comparável). Encha o reservatório com isoflurano e ligue O 2 fluxo de ar. Camundongos Coloque em câmara de indução e ajustar botão de controle de isoflurano a 5% para iniciar a anestesia. Uma vez que os ratos são totalmente anestesiado, indicado pela falta de mobilidade, ajustar botão de controle isoflurano a 3% para manter a anestesia profunda. A finalidade da anestesia neste procedimento é para imobilizar o animal. As injeções de orelha e cauda veia não causar-lhes dor ou sofrimento ao animal. NOTA: O isoflurano pode ter efeitos negativos sobre o sistema reprodutivo humano. Tenha certeza de usar em um quarto bem ventilado e coloque uma lata de eliminação para a câmara de indução para neutralizar gases residuais. Se você está grávida durante o uso de isoflurano, usar uma máscara de filtro de carbono para minimizar a exposição. </li><li> Carregar 10 ml de 5 mg / ml de OVA, para uma seringa de insulina 3/10 cc com uma agulha tampada 31 G. Coloque outro 3/10 cc seringa de insulina com 10 mLPBS 1x. </li><li> Usando um microscópio de dissecação, injectar 10 mL de OVA no ouvido direito de um rato anestesiado por uma dose de desafio de 50 ug de OVA. Outras doses, tais como 20 mg, também são aceitáveis. <ol><li> Para estabilizar o ouvido para injeção, enrole fita adesiva (sticky side up) em torno de um tubo de 15 ml. Fixar o lado dorsal da orelha direita para a fita. Inserir a agulha lado bisel para cima entre as duas folhas da orelha. Lentamente injetar OVA para o centro do tecido da orelha, tomando cuidado para não bater qualquer vasos sanguíneos. </li></ol></li><li> Usando o mesmo método descrito acima, injectam 10 pi de PBS na orelha esquerda como controlo negativo. </li><li> Espere 3 min. <ol><li> Durante este tempo, coloque o mouse em um limitador e segure a cauda sob uma lâmpada de calor ou num banho de água quente para 1 min para dilatar os vasos sanguíneos. </li></ol></li><li> Utilizando uma seringa de insulina de 1 ml tapado com uma agulha 27 G, lentamente injectar 200 ul de 0,5% de corante azul de Evans na cauda vein do mouse. A injecção rápida podem destruir a estrutura do vaso antes de a totalidade do 200 ul é administrado. Se isto ocorrer, a tentativa para injectar o volume restante de corante para dentro da veia no lado oposto da cauda. </li><li> Espere 10 min. </li><li> Eutanásia o mouse e remova ambas as orelhas, usando uma pinça e tesouras cirúrgicas. <ol><li> Agarrar as bordas da orelha com uma pinça, certificando-se de que qualquer pressão é aplicada no local da injecção no centro da orelha. Remover o máximo possível de tecido da orelha de corte perto, mas não através, ao cume de cartilagem presente na base da orelha. Pequenas quantidades de pele pode estar presente no ouvido excisado; isso não afetará o ensaio. </li></ol></li><li> Alíquota de 700 mL de formamida em 1,5 ml microtubos. Prepare um tubo por orelha (dois tubos por mouse). NOTA: Formamide é teratogênico, mutagênico e irritante. Use luvas, proteção para os olhos, e proteção para o rosto. As mulheres grávidas devem ter cuidado ao manusear ums formamida pode ser fetotoxic. </li><li> Adicionar orelhas de formamida, a tampa dos tubos, e incubar durante a noite num banho de calor seco ajustada para 63 ° C. Orelhas ainda pode aparecer um pouco azul após a incubação; no entanto, a maioria do corante será extraído para a formamida. </li><li> Adicionar 300 ul de cada amostra, em duplicado, para uma placa de 96 poços. Evitar a transferência de pele, se estiver presente na amostra. Certifique-se de que não há bolhas presentes nos poços, como elas irão afetar a leitura da absorvância. </li><li> Adicionar 300 ul de formamida, em duplicado, à placa de 96 poços. Estes poços serão utilizados como brancos. </li><li> Utilizar um espectrofotómetro para medir a absorvância a 620 nm. Poços formamida subtrair de cada leitura da amostra. NOTA: Todos os experimentos foram realizados sob protocolos aprovados pelo Comitê Institucional Animal Care e Use a Universidade Militar. </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.

Inúmeros marcadores de doença alérgica são usados ​​para avaliar a força de respostas alérgicas seguintes desafio alérgeno, incluindo alterações nos níveis de histamina circulante, a produção de citocinas Th2, e recrutamento de células no líquido broncoalveolar no cenário de desafio das vias aéreas. Durante a monitorização de alterações nestes parâmetros é importante para o estudo de reações alérgicas, marcadores biológicos nem sempre têm relação com doença alérgica clínica. O ensaio ...

Hipersensibilidade de tipo I é mediada pela induzida por antigénio a ligação cruzada de IgE na superfície dos mastócitos e basófilos. Isso resulta em degranulação celular ea liberação de mediadores vasoativos e pró-inflamatórios como a histamina, triptase e fator ativador de plaquetas 2. Após a liberação de mediadores pré-formados durante a degranulação, mastócitos sintetizam e liberam prostaglandinas e leucotrienos, que aumentam ainda mais a permeabilidade vascular 3. A resposta clínica inicial ocorre rapidamente e é referido como uma &quot;reacção imediata&quot;. Na pele, uma resposta pápula-e-queima é facilmente visível a poucos minutos do desafio antigênico. Dependendo da dose do desafio, é possível observar uma &quot;resposta de fase tardia&quot; algumas horas mais tarde. Inchaço fase tardia é devido a edema localizado e o recrutamento de leucócitos para os tecidos 2. A histamina, geralmente considerado como sendo o mediador principal participa nareações alérgicas imediatas, age em receptores de histamina 1 (HR1), expresso em vasos e receptor de histamina 2 (HR2) expressa no músculo liso. O efeito combinado aumenta o fluxo sanguíneo e da permeabilidade vascular no local da inflamação 4. Uma variedade de modelos animais de alergia têm sido desenvolvidas a fim de estudar os mecanismos envolvidos na inflamação alérgica, incluindo modelos de asma alérgica, anafilaxia sistémica e choque anafilático locais. A administração intravenosa do corante tem sido usado para medir as respostas alérgicas localizadas em modelos animais por quase um século, com publicações que descrevem esta técnica que remonta à década de 1920 5. Os coelhos e as cobaias foram os primeiros modelos animais utilizados para testar as reacções de hipersensibilidade imediata, e as respostas foram mais sensíveis geralmente encontrados no ouvido 5,6. O ensaio foi posteriormente validado para uso em ratos e camundongos 7 8. Historicamente,uma variedade de métodos experimentais têm sido utilizados, incluindo a injecção de antigénio, antes da injecção do corante, a injecção de corante, antes da injecção de antigénio, e a injecção simultânea de corante e antigénio. Administração intravenosa de corante como um meio para medir as respostas alérgicas é um ensaio versátil, uma vez que pode ser utilizado para medir activa, passiva e inverter reacções passivas 5,9. Numerosos corantes têm sido utilizados para avaliar as respostas alérgicas, incluindo Trypan Blue, pontamina Céu Azul, Azul de Evans, Geigy Azul 536, e Índia Ink 5,6,9. Uma solução de 0,5% de azul de Evans é actualmente o corante padrão usado para medir as respostas alérgicas na pele. A resposta anafilática ao desafio é transitório; intensidade máxima é atingida em 10-15 min de injeção de corante, e nenhuma reação é visível se corante é administrada mais de 30 min após o desafio, independentemente das espécies animais utilizados 9. Quantificação de extravasamento de corante foi originally obtida pela medição do tamanho da pápula como indicado pelo corante azul 7-9. Além disso, as contagens de mastócitos desgranulados pode ser quantificado através da excisão de tecido da pele a partir do local da reacção e coloração com azul de toluidina 7. Desgranulação de mastócitos é muitas vezes usada como um marcador para cutânea, as respostas alérgicas mediadas por IgE, como mastócitos são a população principal de células que expressam o local de alta afinidade de IgE do receptor FceRI. Foram desenvolvidas técnicas de espectrofotometria para medir corante extravasamento para o tecido de anafilaxia cutânea passiva (PCA) no rato e rato 10 11 na década de 1990. O seguinte protocolo de ensaio de anafilaxia local, foi adaptado a partir de Kojima et al. 1, e utiliza ovalbumina de ovo de galinha (OVA) como antigénio para provocar respostas alérgicas. No entanto, com excepção de OVA antigénios podem ser utilizadas, se desejado. O ensaio, em seguida, utiliza corante azul de Evans para monitorar as mudanças em permeabilit vasculary que ocorrem devido a mastócitos IgE cross-linking e liberação de histamina.

<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

As reações alérgicas são o resultado da ativação de células e basófilos mastro, ea posterior liberação dos vasoativas e mediadores pró-inflamatórios. A exposição a um alergénio em um indivíduo sensibilizado pode resultar em sintomas clínicos que variam de menor eritema a vida em risco de anafilaxia. No laboratório, vários modelos animais têm sido desenvolvidos para compreender os mecanismos de condução respostas alérgicas. Aqui, nós descrevemos um método detalhado para medir alterações na permeabilidade vascular quantificar respostas alérgicas localizadas. O ensaio de anafilaxia local, foi relatada pela primeira vez em 1920, e foi adaptado a partir da técnica publicada pela Kojima et al. em 2007 1. Neste ensaio, os ratos sensibilizados a OVA são desafiados na orelha esquerda com o veículo e na orelha direita com OVA. Isto é seguido por uma injecção intravenosa de corante azul de Evans. Dez minutos depois da injecção de azul de Evans, o animal é sacrificado e o corante que extravasou empara os ouvidos é extraída durante a noite em formamida. A absorvância do corante extraído é então quantificada com um espectrofotómetro. Este método resulta de forma confiável em uma manifestação visual e quantificável de uma resposta alérgica local.

Os animais que tenham sido submetidos a ensaio com êxito terão os olhos ea pele que aparecem em azul. PBS animais sensibilizados não deve reagir a PBS ou desafio com OVA, portanto, ambos os ouvidos deverá permanecer branco (Figura 1A). Em animais sensibilizados com OVA, na orelha recebendo o desafio PBS (esquerda) deve ser completamente branca ou levemente azul de uma maneira localizada no local da injecção. A orelha recebendo o desafio com OVA (direita) deve tornar-se progressivamente mais escura...

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

Measuring Local Anaphylaxis in Mice

As reações alérgicas, caracterizada pela ativação de mastócitos e basófilos, são movidos pela ligação cruzada de IgE e liberação de mediadores pró-inflamatórios. A avaliação quantitativa da resposta alérgica pode ser conseguido através de corante azul de Evans para monitorar alterações na permeabilidade vascular após o desafio alérgeno.

Medindo Anafilaxia locais em Ratos

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 visualizações.  Uniformed Services University of the Health Sciences.  As reações alérgicas, caracterizada pela ativação de mastócitos e basófilos, são movidos pela ligação cruzada de IgE e liberação de mediadores pró-inflamatórios. A avaliação quantitativa da resposta alérgica pode ser conseguido através de corante azul de Evans para monitorar alterações na permeabilidade vascular após o desafio alérgeno. 

Vídeo: 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.

Medir a atividade das calpaínas em células fixas e Vivendo por citometria de fluxo

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

Imunologia e Infecção

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.

Medir a carga bacteriana e resposta imune em camundongos infectados com 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.

Medição de γHV68 Infecção em Camundongos

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

Intralymphatic Immunotherapy and Vaccination in Mice

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of vaccine to lymph nodes where antigen presenting cells can interact with lymphocytes for generation of the wanted immune responses. The strength and the type of immune responses induced also depend on the density or frequency of interactions as well as the microenvironment, especially the content of cytokines. As only a minute fraction of peripherally injected vaccines reaches the lymph nodes, vaccinations of mice and humans were performed by direct injection of vaccine into inguinal lymph nodes, i.e.&#160;intralymphatic injection. In man, the procedure is guided by ultrasound. In mice, a small (5-10 mm) incision is made in the inguinal region of anesthetized animals, the lymph node is localized and immobilized with forceps, and a volume of 10-20 &#956;l of the vaccine is injected under visual control. The incision is closed with a single stitch using surgical sutures. Mice were vaccinated with plasmid DNA, RNA, peptide, protein, particles, and bacteria as well as adjuvants, and strong improvement of immune responses against all type of vaccines was observed. The intralymphatic method of vaccination is especially appropriate in situations where conventional vaccination produces insufficient immunity or where the amount of available vaccine is limited.

Prophylactic and therapeutic vaccination often fails to stimulate strong immune responses due to week drainage of the vaccine to lymph nodes and consequently poor involvement of immune cells. By direct injection of vaccine to lymph nodes, so-called intralymphatic injection, vaccine efficacy can be strongly improved and vaccine doses can be reduced.

Intralinfática imunoterapia e vacinação em camundongos

Vaccines are typically injected subcutaneously or intramuscularly for stimulation of immune responses. The success of this requires efficient drainage of ...

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.

Medição de deformabilidade e heterogeneidade de glóbulos vermelhos no sangue por Ektacytometry

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.

Caracterização das respostas de isotipo de imunoglobulina Timo-dependentes e Timo-independente nos ratos usando ensaio enzima-lig da imunoabsorção

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.

Medição de temperatura de corpo do mouse usando o termômetro infravermelho durante passiva anafilaxia sistêmica e avaliação de alergia alimentar

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.

Quantificação da aterosclerose em camundongos

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.

Imunoterapia adotiva de células iNKT em Isomerase Isomerase Induzida de Glicose-6-Fosfato (G6PI)-Induzido RaMice

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.

Medição de phagocitose de Aspergillus fumigatus Conidia por Leukocytes Humanos usando citometria flow

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