Os autores agradecem à Universidade de Rochester instalação Multiphoton Microscópio Núcleo de ajuda com imagens ao vivo. Financiado pelo NIH AI072690 e AI02851 para DJF; AI114036 para AG e AI089079 para MGO.

Os autores não têm nada a revelar.

Significado Aqui apresentamos um protocolo completo para a visualização 4D, efetoras antígeno-específica células Th1 transferidos na derme orelha de rato intactas. Este método oferece vantagens em relação a algumas modalidades de imagem corrente para várias razões. Pela imagem da derme da orelha ventral, somos capazes de renunciar a depilação que é necessário para protocolos de imagem envolvendo outros locais da pele. Embora depilatórios são geralmente leves, que foram mostrados para causar o rompimento da barreira da pele 42, um processo que pode estimular uma resposta imune 43,44. Por também evitando procedimentos cirúrgicos invasivos para expor a derme ou hipoderme, este protocolo impede a inflamação induzida por danos e o rápido recrutamento de neutrófilos 37 e outras células do sistema imunológico para a derme. A utilização de um cateter venoso no presente sistema para fornecer anticorpos bloqueadores contra moléculas chave permite a interrogação tempo real do Dynacomportamento mic de células T CD4. A utilização deste protocolo de imagens revelou requisitos críticos para células T CD4 motilidade intersticial 30 que não foram detectados em sistemas in vitro 8. Etapas críticas do processo Um passo essencial em qualquer protocolo de imagem é garantir a preparação do tecido estável para a imagem latente. É importante assegurar que há suficiente PBS entre a orelha e a lamela de que não existem bolhas de ar e a orelha se encontra em contacto com o vidro, mas não tanto como para fazer que a fita de se tornar de-aderiu ao vidro. Do mesmo modo, evitando o contacto entre a fita que prende a lamela para a plataforma e qualquer graxa de vácuo irá evitar que a fita de afrouxamento ao longo do tempo. A temperatura também deve ser mantido constante para evitar oscilações e deriva de contracção ou expansão térmica dos materiais de plataforma. Limitações e Modificações <p clAss = &quot;jove_content&quot;&gt; Imagiologia por este protocolo não invasivo é limitada às áreas da pele que são suficientemente fina para permitir a visualização eficaz de fluorescência através da excitação multifotônica. A orelha é vantajoso devido à facilidade da sua preparação e capacidade de ser isolado a partir de movimentos respiratórios, e tem sido utilizada como um modelo para a imagiologia de lapso de tempo intravital desde a década de 1980 45. No entanto, a população residente imune do ouvido é distinta da pele no flanco da pata ou 46, e a orelha de rato tem propriedades vasculares distintas quando comparado com outros locais 47. Assim, para algumas aplicações, a comparação dos dados de imagem de orelha para outros sítios da pele pode ser difícil. Este protocolo também requer a migração eficaz de células T efectoras transferido para fora da corrente sanguínea e para dentro da derme. Isto limita a possibilidade de usar células que têm defeitos no retorno ou extravasamento uma vez que não será capaz de entrar no espaço intersticial. Extravasamentopode ser ultrapassado através da injecção de células directamente para a derme da orelha 37,48, embora isso irá causar algum dano mecânico e fornecimento de células, desta forma não podem recapitular a localização ou o comportamento de células que sofrem de extravasamento vivo. Também é fundamental considerar o uso de ratos destinatário não pigmentadas para experiências de imagiologia, tais como a estirpe / c BALB utilizado aqui ou ratos Albino C57BL / 6 Tyr c-2J. A melanina em ratos pigmentados, além de ser altamente autofluorescente, aquece, mesmo sob relativamente excitação de baixa potência de um laser multiphoton 37. Isto pode causar danos térmicos à pele e a inflamação subsequente 36 ou fluorescente salpicar 31, complicando resultados. Isto pode limitar fluoróforos que podem ser utilizadas numa experiência de imagem multi-parâmetros. No entanto, algumas moléculas fluorescentes altamente expressos muito brilhantes ou pode ser efetivamente animado a baixa potência do laser, todosdevido para visualização eficaz em ratos pigmentados. As aplicações futuras Porque este é um procedimento não-invasivo, que pode ser facilmente adaptado para estudos longitudinais em camundongos com imagiologia sequencial durante períodos de tempo prolongados. Enquanto marcação fluorescente como descrito aqui iria desaparecer durante períodos de tempo maior do que 3-4 dias, o uso de células endogenamente fluorescentes ou células repórter fluorescentes elimina este problema. Na verdade, nós já usou este protocolo para rastrear em effectors antígeno-específicas vivo- gerados portadores de repórteres de citocinas, incluindo o repórter IFN-y Yeti 30,49 e IL-4 repórter 4get 50. Temos, além disso visualizados células CD4 endógenos em células CD4-Cre EYFP ROSA26-stop-floxed ratos repórter fluorescente 30. Como as propriedades fluorescentes de EYFP e outras proteínas fluorescentes diferem dos corantes químicos como CFSE, podem ser necessárias modificações aos parâmetros de imagempara visualização eficiente. Alterações como o aumento do tempo de permanência do pixel pode aumentar o sinal fluorescente de alguns fluoróforos dim, e diminuindo a duração das imagens de lapso de tempo pode mitigar qualquer fotobranqueamento que pode ser observada. A 900 nm de excitação, não temos observado anteriormente fotobranqueamento significativa de CFSE, CMTMR ou EYFP em intervalos de imagem curtas. Embora este procedimento concentra-se na medição da dinâmica da motilidade das células T CD4 efectora, ela não se limita a esta aplicação. O trabalho actualmente em curso é para medir as interacções dinâmicas de células T efectoras com o antigénio das células através da utilização de ratinhos repórter fluorescentes e injecção de anticorpos conjugados com fluorescência para a derme para marcar células ou estruturas de tecido antes da imagem 51,52. Além disso, enquanto este protocolo demonstra a utilização do cateter de entrega, enquanto anticorpos de bloqueio de imagem, de outros compostos, incluindo inibidores de moléculas pequenas, podeser administrada. Em última análise, este protocolo proporciona uma plataforma flexível para medir dinâmica imunes ao longo do tempo, in vivo, de uma forma não invasiva.

The effector function of CD4 T cells is critically dependent on their ability to rapidly enter and traverse a wide variety of peripheral tissues to survey for damage, locate foci of infection, or cause pathology from chronic infection or autoimmunity. While the processes of homing to inflamed sites1-4 and extravasation5-7 from the vasculature into tissues have been well-characterized, the factors that drive and regulate the interstitial motility of T cells remain undefined. The migration of T cells in complex 3D environments has been studied in vitro through the use of artificial matrices8-10 or microfluidic devices11,12, but these fail to recapitulate the complex and dynamic environment of an in vivo system. It is only recently, with the advent of high-resolution multi-color intravital imaging that it has become possible to study the dynamic behavior of immune cells in situ, allowing for a better understanding of intact immune responses.
Over a decade ago, several influential studies were published that first utilized multiphoton microscopy to address immunological questions. Early studies focused on the behavior of immune cells within explanted lymphoid organs13-16, which were soon followed by techniques to image exposed lymph nodes in anesthetized mice17. Imaging allowed for new fundamental observations about the stages of lymph node priming of T cells18, the mechanisms by which T cells migrate in secondary lymphoid organs19, T cell interactions with other immune cells20,21, and dynamic T cell positioning within the lymph node22. Although many early studies focused on lymph node dynamics, intravital imaging has been since been utilized to image the immune response in many peripheral tissues, including the brain23-25, liver26, lung27, and skin28-30. 
The mouse ear dermis is particularly well poised for imaging, due to the thinness of ear skin, a relative lack of hair, and the ease with which it can be isolated from respiratory movements31. Indeed, the ear dermis has been used to image the interstitial behavior of dendritic cells32,33, T cells28,29,34,35, and neutrophils36,37, and is a well-established site for studying dermal inflammation. Increasingly, non-invasive procedures have been replacing surgical preparations of the skin, including split dermis38,39, flank39,40, or dorsal skin flap window39,41 models, that can induce changes to the local inflammatory milieu. The use of transferred, in vitro-primed, antigen-specific CD4 effector T cells allows for the study of a homogenous population of cells in the context of a dermal inflammatory response30. Here we describe a non-invasive imaging procedure that allows for the visualization of antigen-specific effector CD4 T cells in the dermal interstitium of the inflamed mouse ear, and the ability to manipulate these cells in real-time by introducing blocking antibodies through a venous catheter. We show that this model is effective for tracking the movement of CD4 T cells in the dermis and for querying the mechanisms that govern this motility.

<table border="0" cellpadding="0" cellspacing="0" width="1060">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">BALB/c mice</td>
			<td style="width:145px;">Jackson Laboratories</td>
			<td style="width:196px;">000651</td>
			<td style="width:472px;">Mice used were bred in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">DO11.10 mice</td>
			<td>Jackson Laboratories</td>
			<td>003303</td>
			<td>Mice used were bred in-house</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HBSS</td>
			<td>Fisher</td>
			<td>10-013-CV</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Newborn Calf Serum (NCS)</td>
			<td>Thermo/HyClone</td>
			<td>SH30118.03</td>
			<td>Heat inactivated at 56 &#176;C for 30 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Guinea Pig Complement</td>
			<td>Cedarlane</td>
			<td>CL-5000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-CD8 antibody</td>
			<td>ATCC</td>
			<td>3.155 (ATCC TIB-211)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-MHC Class II antibody</td>
			<td>ATCC</td>
			<td>M5/114.15.2 (ATCC TIB-120)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-CD24 antibody</td>
			<td>ATCC</td>
			<td>J11d.2 (ATCC TIB-183)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-Thy1.2 antibody</td>
			<td>ATCC</td>
			<td>J1j.10 (ATCC TIB-184)</td>
			<td>Antibodies derived from&#160; this hybridoma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Ficoll (Fico/Lite-LM)</td>
			<td>Atlanta Biologicals</td>
			<td>I40650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PBS</td>
			<td>Fisher</td>
			<td>21-040-CV</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">EDTA</td>
			<td>Fisher</td>
			<td>15323591</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">biotinylated anti-CD62L antibody (clone MEL-14)</td>
			<td>BD</td>
			<td>553149</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">streptavidin magnetic separation beads</td>
			<td>Miltenyi</td>
			<td>130-048-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">MACS LS Separation Column</td>
			<td>Miltenyi</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant human IL-2</td>
			<td>Peprotech</td>
			<td>200-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant mouse IL-4</td>
			<td>Peprotech</td>
			<td>214-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">recombinant mouse IL-12</td>
			<td>Peprotech</td>
			<td>210-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-IFNg antibody (clone XMG 1.2)</td>
			<td>eBioscience</td>
			<td>16-7311-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">anti-IL-4 antibody (clone 11b11)</td>
			<td>eBioscience</td>
			<td>16-7041-85</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RPMI</td>
			<td>VWR</td>
			<td>45000-412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Penicillin/Streptomycin</td>
			<td>Fisher</td>
			<td>15303641</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">L-glutamine</td>
			<td>Fisher</td>
			<td>15323671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">2-mercaptoethanol</td>
			<td>Bio-Rad</td>
			<td>161-0710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ovalbumin peptide</td>
			<td>Biopeptide</td>
			<td></td>
			<td>ISQAVHAAHAEINEAGR-OH peptide</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Fetal Calf Serum (FCS)</td>
			<td>Thermo/HyClone</td>
			<td>SV30014.03</td>
			<td>Heat inactivated at 56 &#176;C for 30 minutes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">24-well culture plate</td>
			<td>LPS</td>
			<td>3526</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CFSE</td>
			<td>Life Technologies</td>
			<td>C34554</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">CMTMR</td>
			<td>Life Technologies</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">28 G1/2 insulin syringes, 1ml</td>
			<td>BD</td>
			<td>329420</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">28 G1/2 insulin syringes, 300&#956;l</td>
			<td>BD</td>
			<td>309301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">27 G1/2 TB syringes, 1ml</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">30 G1/2 needles</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">PE-10 medical tubing</td>
			<td>BD</td>
			<td>427400</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">cyanoacrylate veterinary adhesive (Vetbond)</td>
			<td>3M</td>
			<td>1469SB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">heating plate</td>
			<td>WPI</td>
			<td>61830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Heating plate controller</td>
			<td>WPI</td>
			<td>ATC-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Water blanket controller</td>
			<td>Gaymar</td>
			<td>TP500</td>
			<td>No longer in production, newer equivalent available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">water blanket</td>
			<td>Kent Scientific</td>
			<td>TP3E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Isoflurane vaporizer</td>
			<td>LEI Medical</td>
			<td>Isotec 4</td>
			<td>No longer in production, newer equivalent available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isoflurane</td>
			<td>Henry Schein</td>
			<td></td>
			<td>Ordered through Veterinary staff</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">microcentrifuge tubes</td>
			<td>VWR</td>
			<td>20170-038</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">medical tape</td>
			<td>3M</td>
			<td>1538-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">isoflurane nosecone</td>
			<td></td>
			<td></td>
			<td>Built In-house, see Fig 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">imaging platform</td>
			<td></td>
			<td></td>
			<td>Built In-house, see Fig 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">curved forceps</td>
			<td>WPI</td>
			<td>15915-G</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">scissors</td>
			<td>Roboz</td>
			<td>RS-6802</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">glass coverslips</td>
			<td>VWR</td>
			<td></td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">high vacuum grease</td>
			<td>Fisher</td>
			<td>146355D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">cotton swabs</td>
			<td></td>
			<td></td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">delicate task wipes</td>
			<td>Fisher</td>
			<td>34155</td>
			<td>Multiple Equivalent</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:247px;">Olympus Fluoview 1000 AOM-MPM upright microscope with Spectra-Physics MaiTai HP DeepSee Ti:Sa laser</td>
			<td>Olympus</td>
			<td></td>
			<td>call for quote</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">optical table with vibration control</td>
			<td>Newport</td>
			<td></td>
			<td>call for quote</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">25x NA 1.05 water immersion objective for multiphoton imaging</td>
			<td>Olympus</td>
			<td>XLPLN25XWMP2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">objective heater</td>
			<td>Bioptechs</td>
			<td>PN 150815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Detection filter cube</td>
			<td>Olympus</td>
			<td>FV10-MRVGR/XR</td>
			<td>Proprietary cube, can be approximated from individual filters/dichroics</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">anti-integrin &#946;1 antibody (clone hMb1-1)</td>
			<td>eBioscience</td>
			<td>16-0291-85</td>
			<td>Azide free, low endotoxin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">anti-integrin &#946;3 antibody (clone 2C9.G3)</td>
			<td>eBioscience</td>
			<td>16-0611-82</td>
			<td>Azide free, low endotoxin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Texas Red Dextran (70,000 MW)</td>
			<td>Life Technologies</td>
			<td>D-1830</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cd4 t cell interstitial migration in the inflamed dermis

A capacidade das células T CD4 para realizar as funções efectoras é dependente da migração rápida e eficiente destas células em tecidos periféricos inflamado por meio de um mecanismo como-ainda indefinida. A aplicação de microscopia multifotônica para o estudo do sistema imunitário proporciona uma ferramenta para medir a dinâmica das respostas imunitárias em tecidos intactos. Aqui apresentamos um protocolo para a não-invasivo imagiologia multiphoton intravital de células T CD4 na derme orelha de rato inflamadas. Utilização de uma plataforma de imagem personalizada e um cateter venoso permite a visualização dinâmica de células T CD4 + no interstício dérmica, com a capacidade para interrogar estas células em tempo real através da adição de anticorpos bloqueadores de componentes moleculares principais envolvidos na motilidade. Este sistema proporciona vantagens em relação a ambos os modelos in vitro e procedimentos de imagem cirurgicamente invasivos. Compreender os caminhos usados ​​pelas células T CD4 para a mobilidade pode vir a fornecer informações sobre os basic função de células T CD4, assim como a patogénese de ambas as doenças auto-imunes e patologia de infecções crónicas.

A capacidade para estudar as respostas imunes in situ, sem alterar o ambiente imunológico é essencial para estudar as interacções em tempo real das células T efectoras com um tecido inflamado. Imagem da derme da orelha intactas por este protocolo, apresentadas na Figura 1A e B, permite a visualização de células T efetoras fluorescente etiquetado transferidos no interstício dérmica. Isto permite tanto de alta resolução (Figura 1C) e lapso de tempo (Figura 1D, Filme 1) imagens de dinâmica de células T efetoras na derme inflamadas. <img alt="figura 1" src="/files/ftp_upload/53585/53585fig1.jpg" /> Figura 1. alta resolução e 4D imagiologia de células T efetoras no interstício dérmica intacta. (A) esboço Experimental. (B) Fotografia de uma prepar orelhaEd para a imagiologia com o local da emulsão (linha tracejada preta) e a área óptima para imagiologia (linha vermelha) indicado. Projeção (C) Maximal 3D de uma pilha de alta resolução mostrando CFSE marcado Th1 células (verde), segundo sinal harmónico de colágeno fibrilar (azul) e Texas Red-Dextran marcado vasculatura (vermelho). A seta branca indica um de vários folículos pilosos autofluorescentes. Barras de escala representam 50 mm (D) caminhos migratórios de células Th1 rastreados por 30 minutos na derme CFA-inflamados. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Este protocolo de imagem requer a utilização de uma plataforma de imagem especializado que foi construído em casa, bem como um nariz cónico adaptado para a administração de anestesia inalado enquanto imagiologia. A plataforma de imagem (Figura 2A-B) consiste óf uma placa de base de alumínio com uma secção central elevada. Esta seção levantada tem uma porção de inserção alinhada com acrílico sentiu para fornecer suporte para o ouvido sem comprimir e potencialmente danificar o tecido fino. A geometria da nossa configuração necessária a construção de um cone de protecção flexível e ajustável para proporcionar anestesia inalado durante o exame. Este cone de protecção, constituído por um tubo de microcentrífuga modificada ligada a uma secção 50 milímetros de tubo flexível (Figura 2C), está fixada no seu lugar com um suporte composto por tubos de microcentrífuga modificados (Figura 2D) e afixada à plataforma de imagem através de gancho e velcro . O uso de gancho e laço para fecho permite o reposicionamento do conjunto do cone do nariz de modo a que qualquer um dos ouvidos de um rato pode ser trabalhada, e podem ser optimamente posicionados para ratinhos individuais. <img alt="Figura 2" src="/files/ftp_upload/53585/53585fig2.jpg" /> Figura 2. Equipamentos para imagiologia intravital. plataforma de imagem embutido personalizada no topo (A) e vista lateral (B). Nosecone para administração de isoflurano (C) e titular nosecone (D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Cateterização da veia da cauda permite um acesso contínuo à prática para administrar os anticorpos e outras moléculas pequenas que podem difundir para fora da vasculatura, ou moléculas fluorescentes maiores, tais como dextrano elevado peso molecular para identificar vasos sanguíneos. Após a administração de 100 ug de anti-β 1 e anti-integrina β três anticorpos bloqueadores através do cateter, prisão células previamente motilidade no interior da derme (Filme 2). Estas células têm uma velocidade média diminuiu após anticorpo<html> administração (Figura 3A), bem como uma diminuição do índice de meandros, a relação entre o deslocamento total para o comprimento total da faixa (Figura 3B). <img alt="Figura 3" src="/files/ftp_upload/53585/53585fig3.jpg" /> Figura 3. A administração de anti-β 1 e anti-β 3 anticorpos Th1 inibe a migração de células na pele inflamada-CFA. (A) Velocidade média de células Th1 antes e após a administração de 100 ug de anti-β 1 e anti-β 3 integrina anticorpos bloqueadores. (B) índice de Meandering de células Th1, antes e após o bloqueio de anticorpos. Cerca de 100 células rastreadas são a partir de imagens antes e após o bloqueio de anticorpos a partir de um único rato em uma experiência representativa. Estatísticas de Mann Whitney.es / ftp_upload / 53585 / 53585fig3large.jpg &quot;target =&quot; _ blank &quot;&gt; Clique aqui para ver uma versão maior desta figura. Porque o cabelo não é removido da superfície da orelha, os artefactos de imagem do cabelo são comuns. A autofluorescência de folículos pilosos (Figura 4A) e sombreamento e autofluorescência de cabelo sobrejacente (Figura 4B) deve ser evitada sempre que possível, uma vez que podem obscurecer as células T e interferir com o software de análise de imagem automatizado. Do mesmo modo, as bolhas de ar aprisionadas entre a superfície da orelha e a lamela pode levar a artefactos de imagiologia (Figura 4C). A preparação adequada da orelha deve minimizar o número eo tamanho de todas as bolhas restantes. <img alt="Figura 4" src="/files/ftp_upload/53585/53585fig4.jpg" /> Figura 4. Artefatos comuns de autofluorescência de cabelo e pobres pré orelhaparação. folículos pilosos (A) autofluorescente. (B) O cabelo autofluorescente e folículos pilosos (verde) e colágeno (branco) com sombras de cabelo sobrepostas, causando linhas escuras na imagem. (C) Artefato de uma bolha de ar, mostrando a borda dos deslocados, autofluorescentes epiderme queratinizadas (linha pontilhada). Barras de escala representam 50 um. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/53585fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> Além disso, a utilização de múltiplas fontes de calor pode conduzir a problemas com a estabilidade do tecido, devido à expansão e contracção térmicas. Configurações do termostato incorretos, por exemplo, pode levar a grandes oscilações durante o exame que pode tornar a interpretação dos resultados difíceis (Filme 3). É essencial para determinar as configurações ideais para qualquer sistema que fornece uma temperatura constante e maximiza a estabilidade. UltimatEly, uma câmara de imagem de temperatura controlada é a melhor maneira de remover a variabilidade de mudanças na temperatura. <img alt="filme 1" src="/files/ftp_upload/53585/53585movie1.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie1.mov" target="_blank">Filme 1. células Th1 que migram na derme CFA-inflamadas (clique com o botão direito para baixar).</a> Image 30 min time-lapse que mostra células Th1 (verde) que migram na rede de colágeno da derme (segunda geração harmônica, azul). <img alt="Movie 2" src="/files/ftp_upload/53585/53585movie2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie2.mov" target="_blank">Filme 2. Th1 células prisão mediante o bloqueio de β 1 e b 3 integrinas (Botão direito do mouse para baixar).</a> As células (verde) foram fotografadas durante 30 min antes da administração de anticorpos de bloqueio, e em seguida trabalhada durante mais 20 min no mesmo local. <img alt="filme 3" src="/files/ftp_upload/53585/53585movie3.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53585/gaylomovie3.mov" target="_blank">Filme 3. Oscilações de uma placa de aquecimento mal controlada (clique com o botão direito para baixar).</a> Imagem de lapso de tempo de 30 min de Th1 células (verde) e geração de segundo harmônico (azul). Após esta imagem foi recolhida, a placa de aquecimento utilizado foi encontrada para ter um termostato com defeito, causando oscilações na temperatura da plataforma de imagem e subsequente expansão e contracção térmica.

Watch this Scientific Journal Video about Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis at JoVE.com

Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

Migração intersticial imagens de células CD4 T na derme inflamadas

Os mecanismos que regulam a motilidade intersticial de células T efectoras CD4 em locais de inflamação são relativamente desconhecidos. Apresenta-se uma abordagem não invasiva para visualizar e manipular em células T CD4 + in vitro -primed na derme da orelha inflamado, permitindo o estudo do comportamento dinâmico destas células in situ.

The ability of CD4 T cells to carry out effector functions is dependent upon the rapid and efficient migration of these cells in inflamed peripheral ...

imaging-cd4-t-cell-interstitial-migration-in-the-inflamed-dermis

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.6K visualizações.  University of Rochester School of Medicine and Dentistry, Rochester, NY.  Os mecanismos que regulam a motilidade intersticial de células T efectoras CD4 em locais de inflamação são relativamente desconhecidos. Apresenta-se uma abordagem não invasiva para visualizar e manipular em células T CD4 + in vitro -primed na derme da orelha inflamado, permitindo o estudo do comportamento dinâmico destas células in situ. 

Vídeo: Imaging CD4 T Cell Interstitial Migration in the Inflamed Dermis

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

Transdução retroviral de receptor de células T no rato células T

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Imunologia e Infecção

Competitive Homing Assays to Study Gut-tropic T Cell Migration

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial cells and tissue extravasation is a multistep process controlled by different adhesion molecules (homing receptors) expressed on lymphocytes and their respective ligands (addressins) displayed on endothelial cells 1 2. Even though the function of these adhesion receptors can be partially studied ex vivo, the ultimate test for their physiological relevance is to assess their role during in vivo lymphocyte adhesion and migration. Two complementary strategies have been used for this purpose: intravital microscopy (IVM) and homing experiments. Although IVM has been essential to define the precise contribution of specific adhesion receptors during the adhesion cascade in real time and in different tissues, IVM is time consuming and labor intensive, it often requires the development of sophisticated surgical techniques, it needs prior isolation of homogeneous cell populations and it permits the analysis of only one tissue/organ at any given time. By contrast, competitive homing experiments allow the direct and simultaneous comparison in the migration of two (or even more) cell subsets in the same mouse and they also permit the analysis of many tissues and of a high number of cells in the same experiment.
Here we describe the classical competitive homing protocol used to determine the advantage/disadvantage of a given cell type to home to specific tissues as compared to a control cell population. We chose to illustrate the migratory properties of gut-tropic versus non gut-tropic T cells, because the intestinal mucosa is the largest body surface in contact with the external environment and it is also the extra-lymphoid tissue with the best-defined migratory requirements. Moreover, recent work has determined that the vitamin A metabolite all-trans retinoic acid (RA) is the main molecular mechanism responsible for inducing gut-specific adhesion receptors (integrin a4b7and chemokine receptor CCR9) on lymphocytes. Thus, we can readily generate large numbers of gut-tropic and non gut-tropic lymphocytes ex vivoby activating T cells in the presence or absence of RA, respectively, which can be finally used in the competitive homing experiments described here.

Competitive homing experiments allow to directly assessing the migratory properties of two different cell populations in a single mouse. Here we illustrate this procedure by comparing the migration of ex vivo-generated gut-tropic versus non-gut tropic T cells.

Competitiva Ensaios Homing para Estudar Gut trópico-Migração de Células T

In order to exert their function lymphocytes need to leave the blood and migrate into different tissues in the body. Lymphocyte adhesion to endothelial ...

A Quantitative Evaluation of Cell Migration by the Phagokinetic Track Motility Assay

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles1,2,3. Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread4,5. Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell6,7,8. However, many biological questions about cell migration remain unanswered.
The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers.
The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip9,10. With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells11,12, fibroblasts9, neutrophils13, skeletal muscle cells14, keratinocytes15, trophoblasts16, endothelial cells17, and monocytes10,18-22. The protocol involves the creation of slides coated with gold nanoparticles (Au&deg;) that are generated by a reduction of chloroauric acid (Au3+) by sodium citrate. This method was developed by Turkevich et al. in 195123 and then improved in the 1970s by Frens et al.24,25. As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses9,26,27.
In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

The phagokinetic motility track assay is a method used to assess the movement of cells. Specifically, the assay measures chemokinesis (random cell motility) over time in a quantitative manner. The assay takes advantage of the ability of cells to create a measurable track of their movement on colloidal gold-coated coverslips.

Uma avaliação quantitativa da migração celular pelo ensaio Phagokinetic faixa Motilidade

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Em tempo real de imagens ao vivo de células T Sinalização de Formação de Complexos

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Monitoramento da Migração de Células dendríticas usando<sup&gt; 19</sup&gt; F /<sup&gt; 1</sup&gt; H Ressonância Magnética

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Isolation and Th17 Differentiation of Na&iuml;ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-&#946;. After incubation for at least 72 hours and for up to five days at 37 &#176;C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.

Isolation and Th17 Differentiation of Na&#239;ve CD4 T Lymphocytes

With effector functions distinct from other T cell subsets, Th17 cells have been centrally implicated in inflammatory autoimmunity. This in vitro Th17 differentiation protocol provides a means to determine whether na&#239;ve CD4+ T lymphocytes can differentiate into Th17 cells, and to further examine their role in autoimmunity and host response.

Isolamento e Th17 Diferenciação de Linfócitos T CD4 Naïve

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

In Vitro Ensaio para avaliar o impacto da imunorreguladora Pathways on específica para o HIV CD4 T efetoras celular Função

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

O transplante de pele da cauda para estudar as respostas das células T CD4 alogênico em camundongos

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Indução de Murino inflamação intestinal por transferência adotiva de Effector CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Alta</sup&gt; Células T em imunodeficientes Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a na&#239;ve CD4+ T cell. Although the cytokine environment during na&#239;ve T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4+ T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4+ T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of na&#239;ve T cells from mouse and the subsequent steps necessary for polarizing na&#239;ve cells to various T helper effector lineages in vitro.

Mouse Na&#239;ve CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Na&#239;ve CD4+ T cells polarize to various subsets depending on the environment at the time of activation. The differentiation of na&#239;ve CD4+ T cells to various effector subsets can be achieved in vitro through the addition of T cell receptor stimuli and specific cytokine signals.

Rato Naïve CD4<sup&gt; +</sup&gt; Isolamento de células T e<em&gt; In vitro</em&gt; Diferenciação em subpopulações de células T

Antigen inexperienced (na&#239;ve) CD4+ T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition ...