NKS is supported by NIH award K08 AI108690.

Todos os estudos foram conduzidos em análise rigorosa e orientações de acordo com a Comissão dos Assuntos Institucionais animal Cuidado e Uso (IACUC) da Harvard Medical School, que atende aos padrões veterinários estabelecidos pela Associação Americana de Ciência Animal Laboratory (AALAS). 1. Horizontal transmissão de bactérias via co-habitação (Opcional) <ol><li> Para minimizar a contaminação exógena (especialmente se estiver usando camundongos gnotobióticos), praticar uma técnica asséptica durante a montagem gaiolas descartáveis ​​estéreis, utilizando alimentos e água que ambos foram autoclavados. </li><li> Use socos de ouvido ou tags para marcar individualmente 6 semanas de idade C57BL / 6 ratos que abrigam diferentes microbiotas e abrigá-los na mesma gaiola. Dado que os ratos são coprofagia e comer a partir de peletes fecais do chão da gaiola, micróbios irá, naturalmente, ser transmitida horizontalmente entre os ratinhos. </li><li> camundongos co-casa para ≥1 semana para dar tempo para a transferência de microrganismos e mudança imunológica antes da análise. </li></ol&gt; 2. Preparação de uma suspensão de células isoladas a partir da camada intra-epitelial do intestino delgado e Lâmina Própria <ol><li> Preparação de Soluções <ol><li> Prepare meios de extracção (por intestino delgado): 30 ml de RPMI + 93% 5 ul de ditiotreitol (DTT) (v / W) + 60 ul de EDTA 0,5 M + 500 uL de soro fetal de bovino (FBS). Adicione o DTT imediatamente antes da utilização. </li><li> Prepare a mídia de digestão (por intestino delgado): 25 ml RPMI + 12,5 mg dispase + 37,5 mg de colagenase II + 300 ml FBS. Adicione o dispase e colagenase imediatamente antes da utilização. </li><li> Para todas as incubações realizadas a 37 ° C, soluções Pré-aquecer a 37 ° C. </li></ol></li><li> Euthanize o mouse por asfixia com CO2, seguido por deslocamento cervical. </li><li> Coloque o lado o rato dorsal para baixo e pulverizar o abdômen com etanol 70%. Use a tesoura para realizar uma laparotomia cortando sequencialmente a pele e fáscia então peritoneal ao longo da linha média ventral from da sínfise púbica até o processo xifóide, expondo a cavidade peritoneal. </li><li> Utilize uma tesoura para separar o intestino delgado a partir do estômago, ao seccionar o esfíncter pilórico. Remova cuidadosamente o intestino delgado do peritônio, provocando afastado a gordura mesentérica. <ol><li> Para remover completamente o intestino delgado, fazer um segundo corte na junção íleo-cecal. Colocar o intestino delgado isolado em frio (isto é., 4 ° C) RPMI contendo 10% de FBS para maximizar a viabilidade celular. </li></ol></li><li> Gentilmente remover grandes pedaços de gordura usando uma pinça curva. Tenha cuidado para evitar rasgar o próprio tecido intestinal durante a tentativa de remover a gordura. </li><li> Para remover conteúdo intestinal, intestino delicadamente lave com 15 - 20 ml de PBS frio utilizando uma agulha de alimentação 18 G aposta em uma seringa. </li><li> Use a tesoura para extirpar PPs, e colocá-los em RPMI frio contendo 5% de FBS. PPs estão localizados no lado antimesentérica do intestino delgado e aparecem como um branco multi-lobuladamassa. Dependendo da estirpe específica, um rato tem tipicamente 8 - 12 PPs. </li><li> Cortar o intestino delgado em 3 - 4 segmentos polegadas. </li><li> Remover a gordura residual rolando cada segmento do intestino delgado em uma toalha de papel umedecido com RPMI, utilizando um bisturi maçante para provocar a gordura para fora do tecido. </li><li> Rodar o tecido de dentro para fora através da canulação os segmentos intestinais com pinças curvas e agarrar a extremidade distai do tecido. Em seguida, usar um par de pinças consecutivas para remover suavemente o segmento de tecido a partir das pinças curvas (com início na extremidade proximal), resultando no tecido a ser invertido. </li><li> Coloque segmentos de tecido em um copo contendo 30 ml de meio de extracção e uma barra de agitação. Fixar a tampa no copo, e agitar a 500 rpm durante 15 min a 37 ° C; agitação deve ser vigorosa, mas não turbulento. </li><li> Após a incubação, usar um filtro de aço para separar pedaços de tecido a partir do sobrenadante contendo o epitélio, que deve aparecer turva. Este sobrenadante contém intraepithelial linfócitos que pode ser ainda mais analisados, se desejado, ao proceder à filtração da amostra nos passos 2.18 - 2.23. </li><li> agitar manualmente as peças de tecidos em meio RPMI para lavar meios de extracção residuais. </li><li> Coloque o tecido sobre uma toalha de papel seco e lançá-lo sobre a extremidade final várias vezes para facilitar a remoção de muco residuais que não foi libertada por meio de extracção. </li><li> Coloque fragmentos de tecido em um tubo de 1,5 mL com 600 uL de meio de digestão. </li><li> Use a tesoura para picar o tecido até peças não ficar com a tesoura e a solução aparece homogênea. Este passo é crítico para assegurar uma digestão enzimática completa do tecido. </li><li> Adicionar o intestino delgado triturada para um copo contendo 25 ml de meio de digestão. Agita-se a 500 rpm durante 30 min a 37 ° C. No meio da digestão (ie., A 15 min), pipeta cima e para baixo com uma pipeta sorológica para ajudar a quebrar qualquer grandes pedaços de tecido. </li><li> Filtro de tecido digerido (ou epithcamada Elial a partir do passo 2.12, se IELs processamento) através de um filtro de células 100 mm em um tubo de 50 ml. Lavar o filtro com 20 ml de RPMI contendo 10% de FBS. </li><li> Centrifugar a solução filtrada a 500 xg durante 10 min a 4 ° C. </li><li> Cuidadosamente decantar o sobrenadante e ressuspender o sedimento em 1 ml de RPMI contendo 10% de FBS. </li><li> Filtrar células ressuspensas através de um filtro de 40 um da célula em um tubo de 50 ml. Lavar filtro com 20 ml de RPMI contendo 10% de FBS. </li><li> Centrifugar solução filtrada a 500 xg durante 10 min a 4 ° C. </li><li> Cuidadosamente decantar o sobrenadante e ressuspender o pellet em 1 ml de RPMI contendo 2% de FBS. </li></ol> 3. Preparação de uma suspensão de células individuais de placas de Peyer <ol><li> Transferência excisadas PPs a um copo com 25 ml de meio de digestão e uma barra de agitação. Seguro tampa, e centrifugação a 500 rpm durante 10 min a 37 ° C. </li><li> Filtrar PPs digerido através de um filtro de 40 um da célula em um tubo de 50 ml. Se qualquer rema aglomeradosem, pressioná-los através do filtro utilizando a extremidade plana do êmbolo de uma seringa de 1 ml. </li><li> Lavar filtro com 10 ml de RPMI contendo 10% de FBS. </li><li> Centrifugar solução filtrada a 500 xg durante 10 min a 4 ° C. </li><li> Cuidadosamente decantar o sobrenadante e ressuspender o pellet em 1 ml de RPMI contendo 2% de FBS. </li></ol> 4. manchas na superfície de Citometria de Fluxo <ol><li> Aliquota volume apropriado de células (por ex., A 200 ul de LPLs) numa placa de fundo redondo de 96 poços. </li><li> Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar o sobrenadante invertendo a placa. </li><li> Ressuspender as células em 90 jil de meio RPMI contendo FBS a 2% e uma diluição 1: 100 de anti-CD16 / 32 (bloco Fc). Incubar durante 10 min a 4 ° C. </li><li> Adicionar 10 ul de PBS. Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar o sobrenadante invertendo a placa. </li><li> Ressuspender as células em 250 ul de PBS. Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar o sobrenadante atravésinvertendo a placa. </li><li> células Mancha (opcional) com corante de viabilidade, se desejado, seguindo o protocolo do fabricante. </li><li> Ressuspender as células em 100 ul 1% de formalina para fixar as células. Incubar durante 1 hora no escuro a 4 ° C. </li><li> Adicionar 200 ul de PBS. Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar o sobrenadante invertendo a placa. </li><li> Ressuspender as células em 250 ul de PBS. Centrifugar a 500 xg durante 5 min a 4 ° C. Decantar o sobrenadante invertendo a placa. </li><li> Ressuspender as células em 200 ul de PBS. Analise as células utilizando um citómetro de fluxo. </li></ol>

<ol>
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No conflicts of interest declared.

Nós apresentamos um protocolo para o isolamento e caracterização de citometria de fluxo de linfócitos pequenos-intestinal, incluindo a LPLs, IELs e linfócitos no PPs. Para aqueles interessados ​​em avaliar como as mudanças na microbiota afetam o sistema imunológico do intestino delgado, detalhamos os passos simples envolvidos na transmissão horizontal de organismos entre os ratos que abrigam diferentes microbiotas. Embora este protocolo centra-se no intestino delgado, o procedimento é o mesmo para a anális...

A tarefa principal do intestino delgado é muitas vezes considerada a digestão e absorção de nutrientes 1. Enquanto essa função metabólica é claramente essencial, o intestino delgado tem um papel igualmente importante na defesa do hospedeiro, da barragem contínua de antigénios ambientais encontradas no interior do lúmen 2. O tracto intestinal separa o mundo exterior (por exemplo., Antigénios luminais) a partir do ambiente interno do hospedeiro com uma camada epitelial que é apenas uma única camada de células de espessura. Como tal, o sistema imune do intestino delgado tem a tarefa formidável de equilibrar o seu limiar de reactividade, permitindo antigénios estranhos provenientes dos micróbios dieta e comensais de introduzir a mucosa com mínima, se alguma, resposta imune, enquanto a montagem de uma resposta robusta contra patogénios invasores e outras &quot;prejudiciais&quot; antígenos. Respostas imunes excessiva ou inadequada para esses antígenos pode levar a doença patológica (por exemplo., Inflammadoença tory intestino, diabetes tipo I, esclerose múltipla) e deve ser evitado 06/03. No geral, o tracto gastrointestinal é pensado para representar a maior órgão imune no corpo, contendo mais de 70% de todas as células secretoras de anticorpos 7. O sistema imunitário do intestino delgado é constituída por 3 compartimentos principais - a lâmina própria (LP), a camada intraepitelial, e placas de Peyer (PP) - que cada um contém um grupo distinto de linfócitos 2. Os linfócitos LP (LPLs) são principalmente as células TCRαβ + T com ~ células 20% de B; linfócitos intra-epiteliais (IELs) contêm células B muito poucos com mais células TCRγδ + T do que as células TCRαβ + T; e PPS, que são órgãos linfóides secundários embutidos no intestino delgado, parede contêm células ~ 80% de B. Embora cada uma destas regiões anatómicas tem funções ligeiramente distintas e bases ontológicas, elas funcionam em AHmoda armonized para proteger o hospedeiro de insultos patogénicos. Além disso, há um crescente apreciação que a microbiota é um determinante crítico para o desenvolvimento do sistema imunitário intestinal, com o aumento do reconhecimento cognato da relação entre os micróbios e a ontogenia específicos de determinadas linhagens celulares 8,9. Além disso, dado que a formação do sistema imune intestinal afeta as respostas imunes em locais anatomicamente distantes (por ex., Artrite, esclerose múltipla, a pneumonia), tornou-se claro que o desenvolvimento do sistema imunitário do intestino é relevante para mais processos de doença do que anteriormente reconhecido 10 -12. Como tal, o interesse em avaliar quantitativamente o sistema imunológico intestinal foi muito além interações patógeno-hospedeiro para incluir agora interações hospedeiro-comensais e patogênese de muitas doenças sistêmicas bem. Tendo em conta a variabilidade dos métodos actuais naisolamento de linfócitos intestinais, um método que é otimizado para a produção, viabilidade e consistência, equilibrando o tempo necessário é cada vez mais crítica. Os protocolos que envolvem gradientes de Percoll são tempo e trabalho intensivo e potencialmente mais propenso a erro humano, levando a um rendimento variável e viabilidade 13. Aqui, nós fornecemos um protocolo optimizado para o isolamento e caracterização de linfócitos a partir de todos os compartimentos 3 imunes no intestino delgado. Além disso, dado o interesse crescente em alterações induzidas pelo micróbio no sistema imune das mucosas, que incluem passos que podem ser utilizados para permitir a transmissão horizontal de microrganismos entre ratos para avaliar como estas alterações afectam quantitativamente o sistema imune do intestino.

<table>
	<tbody>
		<tr>
			<td>Sterile Gloves</td>
			<td>Kimberly-Clark</td>
			<td>555092</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile mouse cage</td>
			<td>Innovive</td>
			<td>MS2-AD</td>
			<td>contains lid, cage bottom, and alpha-dri bedding</td>
		</tr>
		<tr>
			<td>metal feeder</td>
			<td>Innovive</td>
			<td>M-FEED</td>
			<td></td>
		</tr>
		<tr>
			<td>water bottle</td>
			<td>Innovive</td>
			<td>M-WB-300</td>
			<td></td>
		</tr>
		<tr>
			<td>card holder</td>
			<td>Innovive</td>
			<td>CRD-HLD-H</td>
			<td></td>
		</tr>
		<tr>
			<td>autoclavable rodent chow (NIH-31M)</td>
			<td>Zeigler</td>
			<td>4131207530</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI medium 1640</td>
			<td>Gibco</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)</td>
			<td>Sigma</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA (pH 8.0)</td>
			<td>Ambion</td>
			<td>AM9262</td>
			<td></td>
		</tr>
		<tr>
			<td>fetal bovine serum (FBS)</td>
			<td>GemBio</td>
			<td>100-510</td>
			<td></td>
		</tr>
		<tr>
			<td>dispase II</td>
			<td>Invitrogen</td>
			<td>17105-041</td>
			<td>the concentration in the protocol is based on an activity level of 1.878 U/mg</td>
		</tr>
		<tr>
			<td>collagenase, type II&#160;</td>
			<td>Invitrogen</td>
			<td>17101-015</td>
			<td>the concentration in the protocol is based on an activity level of 245 U/mg</td>
		</tr>
		<tr>
			<td>dissecting scissors</td>
			<td>Roboz</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>feeding needle (18 G, 2&#34; length)</td>
			<td>Roboz</td>
			<td>FN-7905</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml syringe</td>
			<td>BD</td>
			<td>305482</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Scalpel (15 blade)</td>
			<td>Miltex</td>
			<td>4-415</td>
			<td></td>
		</tr>
		<tr>
			<td>curved forceps</td>
			<td>Roboz</td>
			<td>RS-5211</td>
			<td></td>
		</tr>
		<tr>
			<td>straight forceps</td>
			<td>Roboz</td>
			<td>RS-5132</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-purpose cups, 120 ml</td>
			<td>VWR</td>
			<td>89009-662</td>
			<td></td>
		</tr>
		<tr>
			<td>stir bar</td>
			<td>VWR</td>
			<td>58949-062</td>
			<td></td>
		</tr>
		<tr>
			<td>multi-position stir plate, 9-position</td>
			<td>VWR</td>
			<td>12621-048</td>
			<td></td>
		</tr>
		<tr>
			<td>stainless steel conical strainer, 3 inch&#160;</td>
			<td>RSVP</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml tube</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-19</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>08-771-1</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate, round-bottom</td>
			<td>Corning</td>
			<td>3799</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse CD16/32 (Fc block)</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>(optional) fixable viability dye eFluor 780</td>
			<td>eBiosciences</td>
			<td>65-0865-18</td>
			<td></td>
		</tr>
		<tr>
			<td>10% formalin, neutral buffered</td>
			<td>Thermo Scientific</td>
			<td>5725</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and flow cytometric characterization of murine small intestinal lymphocytes

Os intestinos - que contêm o maior número de células imunes de qualquer órgão do corpo - são constantemente expostos a antígenos estranhos, tanto microbiana e dietética. Dado que uma maior compreensão destes antigénios luminais ajudar a dar forma a resposta imune e que a formação de células imunitárias no intestino é crítica para uma série de doenças sistémicas, tem havido um interesse crescente na caracterização do sistema imune do intestino. No entanto, muitos protocolos publicados são árduos e consumidores de tempo. Apresentamos aqui um protocolo simplificado para o isolamento de linfócitos a partir da própria lâmina do intestino delgado, a camada intraepitelial, e placas de Peyer, que é rápido, reprodutível, e não necessita de gradientes de Percoll laboriosos. Embora o protocolo centra-se no intestino delgado, é também adequado para a análise do cólon. Além disso, destacam-se alguns aspectos que podem precisar de otimização adicional, dependendo do ques científica específicação. Esta abordagem resulta no isolamento de um grande número de linfócitos viáveis ​​que podem, subsequentemente, ser utilizadas para a análise de citometria de fluxo ou meio alternativo de caracterização.

Análise citométrica de fluxo de suspensões de células únicas de linfócitos no intestino delgado deve produzir uma população discreta de células que têm características semelhantes dispersão frontal e lateral como esplenócitos (Figuras 1A e 1B). Os linfócitos podem começam a morrer, se o tecido não é mantida a 4 ° C durante as fases iniciais do isolamento, resultando na população de linfócitos com uma dispersão frontal inferior e sendo mais difíceis...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes at JoVE.com

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

Isolamento e Caracterização de citometria de fluxo de linfócitos de murino Intestino Delgado

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

isolation-flow-cytometric-characterization-murine-small-intestinal

Research

JoVE Journal

Immunology and Infection

27.3K visualizações.  Boston Children’s Hospital. There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization. 

Vídeo: Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Isolamento de citometria de fluxo de células primárias Murino Tipo II epiteliais alveolares de Estudos funcionais e Molecular

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Imunologia e Infecção

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

O isolamento de linfócitos de rato Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

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 ...

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolamento e análise de citometria de fluxo de células imunes do cérebro isquêmico Rato

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Isolamento e Análise de Citometria de Fluxo infiltrantes Glioma Células Mononucleares do Sangue Periférico

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

Isolation and Activation of Murine Lymphocytes

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading pathogen1,2. Understandably, such intricate abilities are controlled by a large number of molecules involved in various cellular processes to ensure timely and spatially regulated immune responses3. Here, we describe experimental procedures that allow rapid isolation of highly purified murine lymphocytes using magnetic cell sorting technology. The resulting purified lymphocytes can then be subjected to various in vitro or in vivo functional assays, such as the determination of lymphocyte signaling capacity upon stimulation by immunoblotting4 and the investigation of proliferative abilities by 3H-thymidine incorporation or carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling5-7. In addition to comparing the functional capacities of control and genetically modified lymphocytes, we can also determine the T cell stimulatory capacity of antigen-presenting cells (APCs) in vivo, as shown in our representative results using transplanted CFSE-labeled OT-I T cells.

Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolamento e Activação de Linfócitos murinos

B and T cells, with their extremely diverse antigen-receptor repertoires, have the ability to mount specific immune responses against almost any invading ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Citometria de fluxo de partículas ligadas aos Bet v 1 alérgeno no PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Isolation and Flow Cytometric Analysis of Human Endocervical Gamma Delta T Cells

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore essential for understanding pathogenicity of different pathogens including HIV. Gamma delta (GD) T cells are the prototype of &#39;unconventional&#39; T cells and represent a relatively small subset of T cells defined by their expression of heterodimeric T-cell receptors (TCRs) composed of gamma and delta chains. This sets them apart from the classical and much better known CD4+ helper T cells and CD8+ cytotoxic T cells that are defined by alpha-beta TCRs. GD T cells often show tissue-specific localization and are enriched in epithelium. GD T cells orchestrate immune responses in inflammation, tumor surveillance, infectious disease, and autoimmunity.
Here, we present a method to reproducibly isolate and analyze human endocervical intraepithelial GD T lymphocytes. We have used endocervical cytobrush samples from women participating in the Women&#39;s Interagency HIV Infection Study (WIHS). Knowledge about GD T cells interactions during conditions in which there is an insult to the vaginal mucosal could be applied to any clinical study in which mucosal vulnerability is addressed, including the development of vaginal microbicides.In addition, knowledge about mucosal GD T cell responses has potential for application of GD T cell-based immune therapy in treating infectious diseases.

We describe here an isolation method to obtain human endocervical intraepithelial lymphocytes for the analysis of intraepithelial gamma delta T cells. This protocol can be extended for the purification of endocervical gamma delta T cells by magnetic beads or by cell sorting.

Isolamento e Citometria de Fluxo Analysis of Human Endocervical Gama Cells Delta T

The female reproductive tract (FRT) mucosal immune system serves as the first line of defense. Better knowledge of the genital mucosa is therefore ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

Isolando os linfócitos de rato pequeno Intestinal sistema imunológico

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and their important role in lung development and function, as well as their lung-localized responses to infection and inflammation. To date, no unified method for identification, isolation, and handling of alveolar macrophages from humans and mice exists. Such a method is needed for studies on these important innate immune cells in various experimental settings. The method described here, which can be easily adopted by any laboratory, is a simplified approach to harvesting alveolar macrophages from bronchoalveolar lavage fluid or from lung tissue and maintaining them in vitro. Because alveolar macrophages primarily occur as adherent cells in the alveoli, the focus of this method is on dislodging them prior to harvest and identification. The lung is a highly vascularized organ, and various cell types of myeloid and lymphoid origin inhabit, interact, and are influenced by the lung microenvironment. By using the set of surface markers described here, researchers can easily and unambiguously distinguish alveolar macrophages from other leukocytes, and purify them for downstream applications. The culture method developed herein supports both human and mouse alveolar macrophages for in vitro growth, and is compatible with cellular and molecular studies.

Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages

This communication describes methodologies for isolation and culture of alveolar macrophages from humans and murine models for experimental purposes.

Isolamento e cultura In Vitro de macrófagos alveolares murino e humanos

Alveolar macrophages are terminally differentiated, lung-resident macrophages of prenatal origin. Alveolar macrophages are unique in their long life and ...