O trabalho relatado neste artigo foi apoiado por uma Nacional de Saúde e Pesquisa Médica do Conselho (NHMRC) da Austrália Grant Program (CRP) e um Projeto NHMRC Grant (CRP e BJCQ). BJCQ também é um NHMRC Peter Doherty Fellow Pós-Doutorado.

1) Configuração de reagente 1.1) solução estoque CFSE O corante é CFSE compra diacetato carboxifluoresceína succinimidyl éster (CFDA, SE) (MW 557,47 g / mol), normalmente em 25 frascos mg. Dissolver a 25 mg em 8,96 mL de uma solução de DMSO estoque final de 5 mM (loja como 50-100 mL alíquotas a -20 ° C durante vários meses). CFDA, SE irá reagir com solução aquosa por isso, é fundamental que tal exposição ser evitadas durante o armazenamento. 1.2) Os linfócitos Isolar linfócitos de baço e gânglios linfáticos ou de ratos e ressuspender em 1 ml de meio de cultura (por exemplo, RPMI) com 5% inativado pelo calor (HI) de soro fetal bovino (FCS), com uma concentração celular de 0,5 x 06-10 outubro x 10 7 / mL. 2) Rotulagem CFSE 2.1 Método de rotina): <ol class="lalpha"><li> Completamente ressuspender as células no volume de 1 mL de meio e coloque cuidadosamente no fundo de um fresco (sem contato com o) tubo cônico de 10 mL. </li><li> Coloque o tubo horizontalmente (usando um tubo sem contato com o vai impedir a 1 ml de suspensão de células de se mover e prematuramente mistura com o, CFDA SE soluções). </li><li> Juntar cuidadosamente 110 mL de PBS para a parte não entram em contato com o plástico no topo do tubo que garante que não faz contato com a solução celular. </li><li> Ressuspender 1,1 mL do estoque de 5 mm de CFDA, SE na PBS 110 mL. </li><li> Rapidamente a tampa do tubo e inverter e vortex bem para obter uniforme rápida mistura das soluções. Note-se que o corante CFSE está em uma concentração final de 5 mM, no entanto, a concentração ótima pode ter que ser determinados para cada lote preparado, e então verificado a cada seis meses para garantir que ele é eficaz. </li><li> Incubar as células por 5 minutos em temperatura ambiente. Note-se que após a conversão do CFDA, SE a CFSE (ver discussão) torna-se o corante fluorescente e, portanto, propenso ao branqueamento pela exposição à luz excessiva. Assim, é aconselhável proteger o tubo da luz a partir deste ponto, por exemplo, cobrindo-o com papel alumínio. </li><li> Lave as células através da diluição em 10 volumes de 20 ° C PBS contendo 5% de HI FCS, sedimentando por centrifugação a 300 xg por 5 min a 20 ° C e descartando o sobrenadante. Repita lavar mais duas vezes. </li></ol> 2.2 O método alternativo), que também é adequado para um maior número de células (10 - 30 x 10 7): <ol class="lalpha"><li> Prepare um 2 x (10 mM) CFDA, SE solução pela adição de 2 mL do estoque mM 5-1 mL de PBS e rapidamente adicionar este a 1 mL de células ressuspenso cuidadosamente, em seguida, rapidamente a tampa do tubo e inverter e vortex bem para obter uniforme rápida mistura. </li><li> Incubar as células por 5 min à temperatura ambiente e lave como nos passos 2.1 (f) e 2.1 (g). </li></ol> 2.3) As células podem então ser aplicado a um protocolo de interesse para a indução da divisão celular. Células marcadas pode ser usado em ambos in vitro e em ensaios in vivo. 2.4) Ao término do ensaio de proliferação, as células são colhidas e analisadas por citometria de fluxo (veja abaixo exemplos representativos). 3) Resultados Representante Para avaliar a proliferação de linfócitos por diluição CFSE, é útil conhecer a fluorescência das células indivisível (ou seja, máximo de fluorescência) e não-rotulados células (ou seja, a fluorescência mínima). Portanto, seu projeto experimental deve levar em conta esses controles. Abaixo estão alguns exemplos de ensaios in vitro e em ensaios in vivo utilizando CFSE para medir T CD8 + a divisão celular por citometria de fluxo. 3.1) Em estimulação in vitro A Figura 1 mostra os perfis de CFSE CFSE marcado com ovalbumina (OVA) específicos de células T CD8 + receptor transgênicos OT-I de células T após três dias da cultura com as células dendríticas pulsadas com várias quantidades de OVA. Células T CD8 + purificada a partir do baço e gânglios linfáticos da OT-I ratos foram marcados com CFSE e 1 x 10 5 células cultivadas com 3,3 x 10 4 DC. DC, onde pulsava com OVA por 1 hora a 37 ° C e lavadas duas vezes antes da cultura. Após 3 dias, as células onde colhidas e rotulados com anti-CD8-APC anticorpos e Hoechst 33258 e depois analisados ​​por citometria de fluxo (50 mil eventos coletados). Live (Hoechst 33258 -) CD8 + células são mostrados. Como controles, as células CD8 + T cultivadas por si só, mostra a intensidade CFSE de não-dividida células, enquanto as células não-rotulados mostra a auto-fluorescência das células e os limites de divisões celulares detectáveis. Note-se que quatro picos CFSE pode ser visto em cada um dos painéis, neste exemplo, indicando que as células foram submetidas até 3 divisões. 3.2) Na estimulação vivo A Figura 2 mostra os perfis de CFSE CFSE marcado com receptor de células T OVA-específicas transgênicos CD8 + T OT-I células após 3 dias in vivo in a presença de 20 mg OVA. Células T CD8 + purificada a partir do baço e gânglios linfáticos da CD45.1 congênitas OT-I ratos foram marcados com CFSE e 5 x 10 6 células injetadas iv na veia lateral da cauda C56BL/6J (CD45.2 +) ratos. Depois de 2 hr camundongos foram injetados iv com 20 mg de OVA. Após 3 dias, baços de camundongos foram coletados, feita em uma suspensão de células individuais, rotulada com anti-B220-PerCP, anti-CD8-APC-Cy7 e anti-CD45.1-PE-Cy7 anticorpos e Hoechst 33258 e depois analisados por citometria de fluxo (1.000.000 eventos coletados). Live (Hoechst 33258 -) B220-células são mostrados no painel esquerdo e gated CD8 + + CD45.1 células mostrado no painel direito. Como controles, desestimulado CFSE marcado células T CD8 +, mostra a intensidade CFSE de não-dividida células, enquanto os não-rotulados células mostra a auto-fluorescência das células e os limites de divisões celulares detectáveis. Note que o uso da diferença CD45 allotypic é vital para resolver o CD8 + células T transferidos do host, como eles são um subconjunto menor do total de células CD8 + T (painel esquerdo). Note-se que a maioria das células cair dentro de 7 picos CFSE neste exemplo (painel direito), indicando que as células foram submetidas até 6 divisões. <img src="/files/ftp_upload/2259/2259fig1.jpg" alt="Figura 1" /> Figura 1. Capacidade de CFSE marcado com ovalbumina (OVA) específicos de células T do receptor transgênicos CD8 + células OT-I T para responder in vitro para DC pulsado com diferentes concentrações de OVA, os dados mostraram estar após três dias de cultura. Nota população não-dividida de CD8 + T células na ausência de antígeno (histograma cinza claro) e auto-fluorescência da população não-rotulados (histograma cinza escuro). A figura mostra células T CD8 + que têm dividido 1-3 vezes com base em picos de diluição CFSE, com mais células T dividindo nas concentrações mais elevadas de antígeno. <img src="/files/ftp_upload/2259/2259fig2.jpg" alt="Figura 2" /> Figura 2. Capacidade de CFSE marcado com ovalbumina (OVA) específicos de células T do receptor transgênicos células CD8 + OT-I T, quando adotivamente transferidos em camundongos C57BL / 6 para responder a OVA, os dados apresentados três dias após a transferência adotiva Painel esquerdo:. CD8 +, CD45. 1 + células T OT-1 em ratos destinatário. Painel da direita: perfil proliferação CFSE. Nota da população não-divididas de células T CD8 + em camundongos destinatário não receber o antígeno (histograma cinza claro) e auto-fluorescência de não-rotulados linfócitos (histograma cinza escuro). A figura mostra as células CD8 + T que têm dividido 1-6 vezes com base em picos de diluição CFSE. <img src="/files/ftp_upload/2259/2259fig3.jpg" alt="Figura 3" /> Figura 3. A representação dos vários eventos moleculares que ocorrem durante a marcação das células com diacetato carboxifluoresceína succinimidyl éster (CFDA, SE). Para mais detalhes do processo veja o texto de discussão. Nota: &#39;CFSE&#39; A sigla genérica, não CFDA, SE, é usado para descrever o reagente rotulagem e do processo de rotulagem em geral. Figura baseada em Parish 4.

<ol>
	<li>Lyons, A. B., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+lymphocyte+division+by+flow+cytometry.">Determination of lymphocyte division by flow cytometry.</a> J. Immunol. Methods. 171, 131-137 (1994).</li><li>Parish, C. R., Glidden, M. H., Quah, B. J., Warren, H. S., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+intracellular+fluorescent+dye+CFSE+to+monitor+lymphocyte+migration+and+proliferation.">Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation.</a> Current protocols in immunology. , (2009).</li><li>Quah, B. J., Warren, H. S., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+lymphocyte+proliferation+in+vitro+and+in+vivo+with+the+intracellular+fluorescent+dye+carboxyfluorescein+diacetate+succinimidyl+ester.">Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester.</a> Nature Protocols. 2, 2049-2056 (2007).</li><li>Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+dyes+for+lymphocyte+migration+and+proliferation+studies.">Fluorescent dyes for lymphocyte migration and proliferation studies.</a> Immunol Cell Biol. 77, 499-508 (1999).</li></ol>

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, A fim de apreciar como funciona e porque CFSE rotulagem uniforme, rápido é necessário para seu uso na análise de proliferação, é útil para compreender a base molecular da célula CFSE rotulagem. Isto pode ser dividido em duas fases, 1) entrada de célula e 2) rotulagem Protein (Figura 3). 1) entrada Cell: Entrada do corante para a célula é mediada pela forma diacetilados do corante, diacetato carboxifluoresceína succinimidyl éster (CFDA, SE). Os acetatos fazer a membrana permeante corante altamente permitindo seu fluxo rápido através da membrana plasmática. Esterases, presente dentro da célula, pegará o acetatos de CFDA, SE e, assim, dar lugar à forma CFSE do corante que é permeante de membranas muito menos, portanto, concentrar o corante dentro da célula. 2) rotulagem Proteína: Embora ambos CFDA, SE e CFSE têm amino-reativa cadeias laterais succinimidyl, apenas o CFSE é fluorescente. A alta concentração intracelular de CFSE facilita rotulagem nível rápido e de alta intracelular de proteínas. Rotulagem CFSE de células deve ser realizada rapidamente, a fim de obter uma população homogênea rotulados de células, que é crítico para a resolução de células que sofreram diversas divisões, como mostrado nos resultados representativos (Figura 1 e 2).

<table><tbody><tr><td>CFDA,SE</td><td>Molecular Probes, Life Technologies</td><td></td><td></td></tr><tr><td>DMSO</td><td>Cambridge Isotope Laboratories</td><td></td><td></td></tr><tr><td>Lymphocytes</td><td>eg mouse or human</td><td></td><td></td></tr><tr><td>RPMI</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>FCS</td><td>SAFC Global</td><td></td><td></td></tr><tr><td>10-15ml conical tubes</td><td>Falcon BD</td><td></td><td></td></tr><tr><td>PBS</td><td>GIBCO, by Life Technologies</td><td></td><td></td></tr><tr><td>Aluminium foil</td><td>Capral Aluminium</td><td></td><td></td></tr><tr><td>Vortex</td><td>Scientific Industries Inc.</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Flow cytometer</td><td>BD Biosciences</td><td></td><td></td></tr></tbody></table>

the use of carboxyfluorescein diacetate succinimidyl ester (cfse) to monitor lymphocyte proliferation

Carboxifluoresceína succinimidyl éster (CFSE) é um meio eficaz e popular para monitorar de linfócitos divisão 1-3. CFSE covalentemente rótulos de longa duração de moléculas intracelulares com o corante fluorescente, carboxifluoresceína. Assim, quando uma célula se divide CFSE marcado, a sua progenitura são dotados de metade do número de carboxifluoresceína marcadas moléculas e, assim, cada divisão celular pode ser avaliada medindo a diminuição correspondente na fluorescência celular via citometria de fluxo. A capacidade de CFSE para rotular as populações de linfócitos com uma alta intensidade fluorescente de variância excepcionalmente baixo, juntamente com a sua baixa toxicidade celular, torná-lo um corante ideal para medir a divisão celular. Uma vez que é um corante de fluoresceína baseado também é compatível com uma ampla variedade de fluorocromos outras que a torna aplicável a multi-cor citometria de fluxo. Este artigo descreve os procedimentos normalmente utilizados para a rotulagem linfócitos do mouse com a finalidade de monitorar até 8 divisões celulares. Estas células marcadas pode ser usado tanto para in vitro e in vivo.

Watch this Scientific Journal Video about The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation at JoVE.com

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalentemente rótulos de longa duração de moléculas intracelulares com o corante fluorescente, carboxifluoresceína. Como tal, quando uma célula se divide CFSE marcado, a sua progenitura têm metade da quantidade de fluorescência, que pode assim ser utilizada para avaliar a divisão celular. Este artigo descreve os procedimentos normalmente utilizados para a rotulagem linfócitos mouse com CFSE.

O Uso de diacetato carboxifluoresceína Succinimidyl Ester (CFSE) para monitorar a proliferação de linfócitos

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

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Research

JoVE Journal

Immunology and Infection

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

91.7K visualizações.  John Curtin School of Medical Research, Australian National University. CFSE covalentemente rótulos de longa duração de moléculas intracelulares com o corante fluorescente, carboxifluoresceína. Como tal, quando uma célula se divide CFSE marcado, a sua progenitura têm metade da quantidade de fluorescência, que pode assim ser utilizada para avaliar a divisão celular. Este artigo descreve os procedimentos normalmente utilizados para a rotulagem linfócitos mouse com CFSE.

Vídeo: The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation. This labeling has classically been used to study proliferation and migration. In the method presented here, we have shortened the timeline after adoptive transfer to look at survival and killing of epitope specific CFSE labeled target cells4-6. The level of specific killing of a CD8 + T cell clone can indicate the quality of the response, as their quantity may be misleading. Specific CD8+ T cells can become functionally exhausted over time with a decline in cytokine production and killing7,8. Also, certain CD8 + T cell clones may not kill as well as others with differing TCR specificities9. For effective Cell Mediated Immunity (CMI), antigens must be identified that produce not only adequate numbers of responding T cells, but also functionally robust responding T cells. Here we assess the percent cell specific killing of two peptide specific T cell clones in BALB/c mice.

Antigen Specific In Vivo Killing Assay using CFSE Labeled Target Cells

Many infections elicit a strong CTL response, but occasionally, the quantity of responding cells does not correlate to control of the pathogen1. One measure of CTL quality is their ability to kill specifically2. CFSE labeling of target cells can be used to investigate this CTL response quality in vivo3,4.

Specific Antigen In Vivo Ensaio Killing usando células CFSE rotuladas Alvo

Carboxyfluorescein diacetate succinimidyl ester (CFSE) can be used to easily and quickly label a cell population of interest for in vivo investigation.  ...

Imunologia e Infecção

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Identificação e isolamento de células em divisão lento no glioblastoma humano usando Carboxifluoresceína Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Medicina

Cell-based Flow Cytometry Assay to Measure Cytotoxic Activity

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells to kill CD4+ T cells loaded with their cognate peptide. Target CD4+ T cells are divided into two populations, labeled with two different concentrations of CFSE. One population is pulsed with the peptide of interest (CFSE-low) while the other remains un-pulsed (CFSE-high). Pulsed and un-pulsed CD4+ T cells are mixed at an equal ratio and incubated with an increasing number of purified CD8+ T cells. The specific killing of autologous target CD4+ T cells is analyzed by flow cytometry after coculture with CD8+ T cells containing the antigen-specific effector CD8+ T cells detected by peptide/MHCI tetramer staining. The specific lysis of target CD4+ T cells measured at different effector versus target ratios, allows for the calculation of lytic units, LU30/106 cells. This simple and straightforward assay allows for the accurate measurement of the intrinsic capacity of CD8+ T cells to kill target CD4+ T cells.

This protocol describes a sensitive, cell-based cytotoxicity assay. By enumerating the decrease in frequency of live target CD4+ T cells in the presence of an increasing number of effector CD8+ T cells, this assay allows for the direct assessment of cytolytic activity of antigen-specific CD8+ T cells.

Baseada em células de Citometria de Fluxo Ensaio medir citotóxicos Atividade

Cytolytic activity of CD8+ T cells is rarely evaluated. We describe here a new cell-based assay to measure the capacity of antigen-specific CD8+ T cells ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8+ T proliferation and to promote CD4+Foxp3+ Treg expansion in vitro.

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Caracterização funcional de macrófagos regulatórios que inibem a imunidade reativa ao enxerto

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

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 ...

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol specifically intended to measure antigen-specific killing without an infection. This protocol is designed and describes methods to overcome these limitations by allowing for the detection of antigen-specific killing of a target cell by CD8+ T cells in vivo. This is accomplished by merging a vaccination model with a traditional CFSE-labeled target killing assay. This combination allows the researcher to assess the antigen-specific CTL potential directly and quickly as the assay is not dependent upon tumor growth or infection. In addition, the readout is based on flow cytometry and so should be readily accessible to most researchers. The major limitation of the study is identifying the timeline in vivo that is appropriate to the hypothesis being tested. Variations in antigen strength and mutations in the T cells that may result in differential cytolytic function need to be carefully assessed to determine the optimal time for cell harvest and assessment. The appropriate concentration of peptide for vaccination has been optimized for hgp10025-33 and OVA257-264, but further validation would be needed for other peptides that may be more appropriate to a given study. Overall, this protocol allows a quick assessment of killing function in vivo and can be adapted to any given antigen.

In Vivo Assay for Detection of Antigen-specific T-cell Cytolytic Function Using a Vaccination Model

The goal of this protocol is to allow for detection of in vivo antigen-specific killing of a target cell in a murine model.

Na Vivo Ensaio para detecção de células T antígeno-específicas função citolítica usando um modelo de vacinação

Current methodologies for antigen-specific killing are limited to in vitro use or utilized in infectious disease models. However, there is not a protocol ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

Adaptando os ensaios de citotoxicidade in vivo para estudar Imunodominância em respostas de células T CD8+ específicas do tumor

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear cells (PBMCs). The development of stable, non-toxic, fluorescent dyes such as carboxyfluorescein succinimidyl ester (CFSE) allows for rare, antigen-specific T cells to be distinguished from bystanders by diminution in fluorescent staining, as detected by flow cytometry. This method has the following advantages over alternative approaches: (i) it is very sensitive to low-frequency T cells, (ii) no knowledge of the antigen or epitope is required, (iii) the phenotype of the responding cells can be analyzed, and (iv) viable, responding cells can be sorted and used for further analysis, such as T cell cloning.

Quantification of Proliferating Human Antigen-specific CD4+ T Cells using Carboxyfluorescein Succinimidyl Ester

Presented here is a protocol for measuring proliferating CD4+ T cells in response to antigenic proteins or peptides using dye dilution. This assay is particularly sensitive to rare antigen-specific T cells and can be modified to facilitate cloning of antigen-specific cells.

Quantificação de células T antígeno-específicas humanas proliferating de CD4+ usando o éster do Succinimidyl do carboxyfluorescein

Described is a simple, in vitro, dye dilution-based method for measuring antigen-specific CD4+ T cell proliferation in human peripheral blood mononuclear ...

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation After Stimulation Using CFSE Staining and Flow Cytometry

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3 
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur Institute, Paris, France 
2 INSERM U1223, Paris, France 
3 Universit&#233; Paris Diderot, Sorbonne Paris Cit&#233;, Cellule Pasteur, Paris, France 
4 Flow Cytometry Platfrom, Cytometry and Biomarkers UtechS, Center for Translational Science, Pasteur Institute, Paris, France
The cell cycle is a universal process of life. During the cell cycle, a cell undergoes several modifications to divide into two daughter cells. This mechanism occurs throughout an organism&#39;s life in response to its needs. Cell divisions and embryonic development produce a full organism from a single-celled zygote. During adulthood, the cell cycle is central to many critical biological processes, such as tissue repairs.
Mechanisms of cell division are tightly controlled events where the cell undergoes stepwise modifications before final division. Cells that are not yet in the cycle are described as being in the Gap 0 (G0) phase. During this stage the cell is considered quiescent. When the cell starts to cycle, four distinct phases are recognized: Gap 1 (G1), Synthesis (S), Gap 2 (G2) and Mitosis (M). G1 phase is a checkpoint for resources needed by the cell for DNA synthesis. Then, S phase occurs, and DNA replication starts, followed by the G2 interphase, another checkpoint that controls all elements necessary to for the cell to divide. Finally, the cell enters mitosis and divides into two daughter cells.
Cell division is a highly informative parameter in many different biological systems. In the field of immunology, analysis of leukocyte proliferation can indicate the mechanism of the immune response. Other domains of investigation also rely on cell cycle analysis. For instance, analysis of the cell cycle during tumor development has improved our understanding of cancer.
Many fluorescent dyes are now available for tracking cell proliferation. These dyes differ in their chemical and spectral properties. Two different classes of dyes exist: protein dyes permanently combine with protein by forming a covalent bond, and membrane dyes stably intercalate within cell membranes via strong hydrophobic associations. In vitro and in vivo studies of immune cell proliferation by flow cytometry are among the most common applications of both classes of cell tracking dyes (1, 2).
CFSE (Carboxyfluorescein succinimidyl ester) is a fluorescent dye that marks dividing cells. Initially, all the cells receive the same amount of dye; dividing cells evenly split the dye they received between their two daughter cells. Consequently, the cell cycle can be followed by the progressive decrease of dye intensity in the cells. CFSE staining is followed by conventional multi-parametric flow cytometry, a high-throughput, fluorescence-based technology that allows phenotypic and functional characterization of cells based on their degree of CFSE staining (3).
In the following experiment, we assess the proliferation of CD4+ and CD8+ T cells in-vitro, following CD3 stimulation, using CFSE staining and flow cytometry.

Cell Cycle Analysis: Assessing CD4 and CD8 T Cell Proliferation

Análise do Ciclo Celular: Avaliação da Proliferação de Células T CD4 e CD8 Após Estimulação Usando Coloração CFSE e Citometria de Fluxo

Source: Perchet Thibaut1,2,3, Meunier Sylvain1,2,3, Sophie Novault4, Rachel Golub1,2,3
1 Unit for Lymphpoiesis, Department of Immunology, Pasteur ...

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An In Vitro Assay for Evaluating the Immunomodulatory Impact of Monocytes on Leukocyte Proliferation

Source: Hsu, P., et al. <a href="https://app.jove.com/v/53265">Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs).</a> J. Vis. Exp. (2015)
This video demonstrates an in vitro assay to explore the immunomodulatory influence of mesenchymal stem cells (MSCs) on effector T cell proliferation. The assay includes co-culturing MSCs with PBMCs to induce the monocyte population to acquire an immunomodulatory phenotype, leading to the secretion of interleukin-10 (IL-10). Following incubation with activated CD4+ T cells, the induced monocytes effectively suppress T cell proliferation.

Um ensaio in vitro para avaliar o impacto imunomodulador dos monócitos na proliferação de leucócitos

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Density Gradient Isolation of ...

O Uso de diacetato carboxifluoresceína Succinimidyl Ester (CFSE) para monitorar a proliferação de linfócitos

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