Name: isolation and activation of murine lymphocytes
Uploaded: 2016-10-30T14:00:00.000+00:00
Duration: 8 min 8 s
Description: Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

O estudo é apoiado pelo Ministério da Educação, Singapore (ACRF Tier1-RG40 / 13 e Tier2-MOE2013-T2-2-038). O manuscrito foi editado por Amy Sullivan de Obrizus Communications.

Todos os ratos são criados e mantidos sob condições específicas livres de patógenos e todos os protocolos do mouse são conduzidos de acordo com as diretrizes da Comissão Cuidado e Uso Institucional Animal. 1. Preparação de Tampões e reagentes <ol><li> Prepare completa do Roswell Park Memorial Institute (RPMI) meio (10% de soro inactivado por calor de bovino fetal (FBS), 2 mM de L-glutamina, penicilina (100 UI / mL) / estreptomicina (100 ug / ml), 55 uM de 2-mercaptoetanol ). </li><li> Prepare 20x solução salina equilibrada (BSS) Stock 1 e da 2, separadamente. <ol><li> Prepare 20x BSS Estoque 1 (111 mM dextrose, fosfato de potássio 8,8 mM, 26,7 mM fosfato de sódio dibásico em 1 L de água esterilizada) e adicionar 40 ml de 0,5% Fenol Vermelho 20x estoque BSS 1 antes do ajuste de volume final. Filtrar em condições estéreis através de filtro de 0,2 um antes de armazenar a 4 ° C. </li><li> Prepare 20x BSS stock 2 (25,8 mM de cloreto de cálcio di-hidratado, cloreto de potássio 107 mM, ch de sódio 2,73 Mloride, 19,6 mM de cloreto de magnésio hexa-hidratado, sulfato de magnésio 16,6 mM em 1 L de água estéril). Filtrar em condições estéreis através de filtro de 0,2 um antes de armazenar a 4 ° C. </li><li> Para preparar 1x BSS para uso experimental, diluir 50 ml BSS Estoque 1 e 50 ml da BSS 2, separadamente, em 400 ml de água estéril cada. Combinar as duas soluções diluídas, ajustar até pH 7, e adicionar 20 ml de FBS (2%). Topo até 1 L com água estéril e filtro estéril usando 0,2 m de filtro. NOTA: soluções de BSS devem ser preparados separadamente, pois a mistura de stocks BSS concentradas diretamente pode resultar em precipitação. </li></ol></li><li> Glóbulo Vermelho (RBC) Tampão de Lise <ol><li> Para preparar tampão de lise dos glóbulos vermelhos, mistura 9 partes de cloreto de estoque de amónio (cloreto de amónio 155 mM em água esterilizada) para uma parte da base Tris (130 mM de Tris (hidroximetil) -aminometano em água estéril, pH 7,65) antes do uso. NOTA: Loja soluções de estéreis de cloreto de amônio e Tris-base a 4 ° C. Prepare lise bpode ser prejudicado fresco para assegurar a lise eficiente de hemácias. </li></ol></li></ol> 2. Geração de Linfócitos suspensão de baço ou gânglios linfáticos NOTA: É importante para preparar todos os reagentes e equipamentos necessários para a experiência antes da eutanásia do rato e para gerar suspensões de células únicas de linfócitos tão rapidamente quanto possível para manter as viabilidades celulares elevadas. <ol><li> Euthanize rato experimental por deslocamento cervical ou CO 2 asfixia. NOTA: A partir desta etapa em diante, todos os procedimentos experimentais devem ser realizados de forma asséptica. </li><li> Mergulhe o rato inteiro em 70% de etanol antes de fazer qualquer incisões. Remover os gânglios linfáticos e do baço assepticamente 8, e colocá-los em separadas tubos de 15 ml contendo 5 ml de gelo-frio RPMI / FBS (meio RPMI com SFB a 2%) ou BSS / FBS (a partir do passo 1.2.3). NOTA: Uma vez que BSS impede eficiente lise RBC, use RPMI para a preparação da suspensão de células do baço e mudar para BSS aftelise r RBC. </li><li> Para gerar uma suspensão de células isoladas a partir do baço ou nodos linfáticos, colocar o órgão (s) entre duas peças de estéril de malha 100 uM coador de células numa placa de Petri contendo 2 ml de gelo-frio RPMI / FBS ou BSS / FBS. Usando o êmbolo de uma seringa de 1 ml, triturar o órgão (s) até que tenha sido rasgada em partes muito finas. </li><li> Transferir a suspensão de células para um tubo de 15 ml e lava-se o filtro de células de malha com gelada RPMI / FBS ou BSS / FBS. Recolhe-se o restante suspensão de células, adicioná-lo para o mesmo tubo de 15 ml, e girar a 453 xg durante 5 min a 4 ° C. Remover o sobrenadante. NOTA: Re-suspender as células peletizadas sacudindo o tubo com os dedos antes de adicionar tampão de lise RBC ou médio nas etapas subsequentes. </li><li> Preparar tampão de lise temperatura ambiente (RT) RBC durante a centrifugação da suspensão de células (ver passo 1.3.). Após a peletização das células e remoção do sobrenadante, re-suspender as células com 1 ml de tampão de lise dos glóbulos vermelhos para cada 10 8 células. incuBATE a reacção de lise à TA durante 3-4 min. </li><li> Pare a lise de RBC com 14 ml de gelo-BSS frio / FBS e girar a 453 xg durante 5 min a 4 ° C. </li></ol> 3. Purificação de células B e T <ol><li> A purificação de células B <ol><li> Contar as células usando um hemocitômetro. Re-suspender até 10 8 células esplênicas em 300 ul BSS / FBS e adicionar 50 ul anti-CD43 microesferas magnéticas 9,10. Para remover as células mortas, adicionar 30 ul anexina V esferas magnéticas. Incubar a suspensão de células num frigorífico a 4 ° C durante 30 min. </li></ol></li><li> Purificação de células T <ol><li> Contar as células usando um hemocitômetro. Re-suspender-se a 10 8 células em não-T cocktail de anticorpos de depleção de células (anticorpos biotinilados contra CD19, B220, Gr-1, TCR-γδ, CD49b, CD11c, CD11b, Ter119 e CD4 ou CD8, dependendo da população de células alvo para ser purificado), diluído 1: 200 em 200 ul BSS / FBS a 4,5. Incubar a suspensão de células numa4 ° C frigorífico durante 15 min. </li><li> Após incubação, adicionam-se 10 ml de BSS / FBS para lavar as células e girar a 453 xg durante 5 min a 4 ° C. Remover o sobrenadante e ressuspender as células em 165 ul de BSS / FBS a 30 com micro-esferas de estreptavidina ul e 15 ul anexina V esferas magnéticas. Incubar a suspensão de células num frigorífico a 4 ° C durante 30 min. NOTA: Para assegurar mesmo marcação com as microesferas magnéticas, incubar as células com microesferas durante 15 minutos, em seguida, misturar a suspensão de células suavemente batendo no tubo de 15 ml e incubar mais 15 minutos durante o passo 3.1.1. ou 3.2.2. </li></ol></li><li> Preparação da coluna de separação para a purificação celular <ol><li> Prepara-se uma coluna de separação não utilizada, durante a etiquetagem micropérola das células (passo 3.1.1. Ou 3.2.2.). Pré-aquecido BSS (sem FBS) à temperatura ambiente e utilizar 2 ml de lavar e equilibrar a coluna em condições assépticas. Após o equilíbrio com BSS, a coluna lavada não devem ser deixadas a secar fora. NOTA: Nós usamos os LS column em vez da coluna LD recomendada, devido à sua possibilidade de reutilização (ver passo 3.4.). </li><li> Após marcação com as esferas magnéticas, adicionar 14 ml de BSS / FBS para lavar as células e girar a 453 xg durante 5 min a 4 ° C. Remover o sobrenadante e re-suspender as células em 1-3 ml RT BSS. </li><li> Anexar uma agulha estéril 21 L para a ponta da coluna de reduzir a taxa de fluxo durante o processo de purificação. Carregar a suspensão de células na coluna equilibrada e recolher o fluxo através contendo as células alvo purificados. Nota: Evite a introdução de bolhas na coluna durante o carregamento. </li><li> Lava-se a coluna uma vez com 1 ml de BSS / FBS e recolher o fluxo através contendo as células alvo purificados. Recarregar a coluna com o fluxo através de uma vez. Recolhe-se o fluxo através após a segunda carga no mesmo tubo de 15 ml. </li><li> Lavar a coluna 3 vezes com 1 ml de BSS / FBS e recolher o fluxo através contendo as células alvo purificados. Depois disso, adicionar 5 ml de BSS / FBS a the coluna e, com um êmbolo, lave as células magneticamente marcadas para fora da coluna para um novo tubo de 15 ml. </li><li> Verifique a pureza das células recolhidas por citometria de fluxo utilizando anticorpos que se ligam a antigénios purificados de células T 4,5 B ou superfície. </li></ol></li><li> Re-usando a coluna de separação NOTA: A coluna LS pode ser reutilizado até 4 vezes sem afectar a eficiência da purificação. <ol><li> Lavar a coluna 3 vezes com 5 ml de solução salina tamponada com fosfato (PBS) e 3 vezes com 5 ml de água destilada a partir do topo usando o êmbolo. </li><li> Lavar a coluna com 5 ml de etanol a 70% e seca-se a coluna extensivamente usando uma torneira de ar para evitar a formação de ferrugem na coluna. </li><li> Para preparar uma coluna LS usado para uma experiência de purificação separada, lavar a coluna de baixo para cima com 5 ml de etanol a 70% utilizando o adaptador de seringa. Em seguida, lava-se a coluna a partir de baixo para cima duas vezes com 5 ml de PBS estéril, seguido de 5 ml de PBS, uma vez a partir do topo da coluna. Adicionar 2ml RT BSS para equilibrar a coluna e depois prosseguir para carregar a coluna com células marcadas. </li></ol></li></ol> 4. CFSE Labeling e Estimulação NOTA: Purificado células podem ser submetidas a uma variedade de ensaios in vitro e em ensaios funcionais in vivo. Aqui, utilizamos células T purificadas para determinar a capacidade de estimulação de célula T de APCs 5. <ol><li> solução pré-aquecido rotulagem (0,1% de FBS em PBS) a 37 ° C antes do carregamento CFSE. NOTA: Usando uma baixa percentagem de FBS em PBS reduz a morte celular durante CFSE carga e minimiza a perda de células durante a centrifugação. No entanto, muito FBS pode interferir com carga CFSE. </li><li> Lavar as células purificadas duas vezes com solução de rotulagem, em seguida, re-suspensas a 2 x 10 7 células / ml na solução de marcação pré-aquecido em um tubo de 15 ml. </li><li> Preparar solução de 10 uM CFSE (diluição 1: 500 de CFSE 5 mM de solução de reserva) em solução de etiquetagem pré-aquecido. solução CFSE deveser preparada a cada vez para alcançar rotulagem ideal. </li><li> Para carregar as células com CFSE, adicionar uma suspensão de células parte para 1 parte de solução de 10 uM CFSE em um tubo de 15 ml e incuba-se no escuro durante 10 min a 37 ° C. Uma concentração final de 5 uM CFSE é utilizado para marcar 1 x 10 7 células / ml. </li><li> Inverter o tubo a cada dois minutos para assegurar uma mistura homogénea de células durante o carregamento CFSE. </li><li> Para parar a reacção, adicionar vários volumes de meio RPMI completo gelada e girar a 453 xg durante 5 min a 4 ° C. Após o bem sucedido CFSE carregamento, o sedimento celular aparece amarelado. </li><li> Lavar as células CFSE carregado mais uma vez com meio RPMI completo gelada e girar a 453 xg durante 5 min a 4 ° C antes de utilizar para a cultura in vitro ou in vivo estimulação. </li></ol> 5. estimulação in vitro Em <ol><li> Preparar uma solução 2x estoque de estímulos (2x estímulos solução estoque) imediatamente antes da utilização de modo a que 100 ul de solução de estoque 2x estímulos pode ser adicionada a 100 ul de células até um volume final de 200 ul por poço numa placa de 96 poços. </li><li> Se é necessária uma placa revestida com estímulos (IgM ou CD40 para células B ou CD3 e CD28 para as células T), dilui-se os estímulos em PBS e pré-revestimento da placa de cultura a 4 ° C durante a noite. Em alternativa, a placa de cultura podem ser revestidos a 37 ° C durante 1 hora no dia da experiência. Lave a placa revestida duas vezes com PBS (Não permita que o prato para secar a qualquer momento). </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Para as células B </td><td></td></tr><tr><td> estímulos </td><td> A concentração final </td></tr><tr><td> F (ab &#39;) 2 de cabra anti-IgM de ratinho </td><td> 0,6-2,4 ug / ml </td></tr><tr><td> Anti-ratinho CD40 mAb </td><td> 0,5-2 ug / ml </td></tr><tr><td> recombinanterato IL-4 </td><td> 25 U / ml </td></tr><tr><td> lipopolissacarídeo </td><td rowspan="2"> 0,1-10 ug / ml </td></tr><tr><td> (LPS) de E. coli serotipo 055: B5 </td></tr><tr><td></td><td></td></tr><tr><td> Para as células T </td><td></td></tr><tr><td> estímulos </td><td> A concentração final </td></tr><tr><td> Anti-CD3 (placa revestida) </td><td> 2-10 ug / ml </td></tr><tr><td> (50 ul / poço para o revestimento) </td><td></td></tr><tr><td> Anti-CD28 (placa revestida) </td><td> 2 ug / mL </td></tr><tr><td> IL-2 recombinante </td><td> 40 U / ml </td></tr><tr><td> PDBu (éster de forbol) </td><td> 5-50 ng / ml </td></tr><tr><td> A23187 (ionóforo de cálcio) </td><td> 250 ng / mL </td></tr></tbody></table> Quadro 1: Concentrações de estímulos utilizados para estimular os linfócitos em cultura in vitro.<ol start="3"><li> Para as células B CFSE-rotulados, re-suspender a 3 X 10 6 células / ml em meio RPMI completo e a cultura em triplicado com 3 x 10 5 células / poço em placas de 96 poços de fundo plano, durante 72 h. </li><li> No caso de células T marcadas com CFSE-, re-suspender a 0,5-3 x 10 6 células / ml em meio RPMI completo e a cultura em triplicado com 0,5-3 x 10 5 células / poço em placas de 96 poços de fundo redondo de 48 ou 72 hr . </li></ol> 6. Promoção Vivo Em <ol><li> Para a estimulação in vivo, transferência adoptiva 4 x 10 6 células T CFSE marcados por ratinho (por via intravenosa (IV) em 200 ul de PBS) em cada rato receptor semelhantes em MHC. NOTA: Neste protocolo, as células T CFSE rotulados podem ser adoptively transferidos usando veia da cauda ou injeção retro-orbital como essas células vão para casa para linfóides órgãos como o baço e os linfonodos. </li><li> Desafiar os ratos receptores um dia mais tarde com o antigénio. NOTA:Neste exemplo, usamos a ovalbumina (OVA proteína, 50 ug / ratinho) como antigénio específico porque OVA-, receptor de células T (TCR), as células T foram -transgenic adoptivamente transferidos para ratinhos receptores. Prepare proteína OVA em PBS estéril e injectar 100 ul de proteína OVA / PBS ou PBS de controlo, por meio de injecção subcutânea (Si), em cada rato receptor 5. </li><li> Colheita e gerar suspensões individuais de células de órgãos linfóides (nódulos linfáticos e baços de ratinhos receptores) 3 dias após a imunização com a proteína OVA ou PBS. linfonodos separar em nodos linfáticos proximais (PLN), que inclui axilar, braquial e nódulos linfáticos cervical superficial) e nódulos linfáticos distais (DLN), que inclui mesentérica, popliteal, inguinal, lombar, e nódulos linfáticos caudal. células mancha usando os anticorpos FACS apropriados para verificar se há a proliferação de células T. NOTA: Nesta célula experimento de rastreamento CFSE, CFSE marcado com células T de ratinhos de controlo injectados com PBS estabelecer uma linha de base para a fluorescência não-dinecer células. Divisões celulares de proliferação, as células, marcadas com CFSE-estimulado com antigénio são visualizados através da medição de fluorescência picos 12,13. Com o controlo de PBS, o número de divisões celulares de proliferação, as células T CFSE-marcados podem ser determinadas 12,13. </li><li> Analisar os dados de proliferação de células marcadas com CFSE, comparando o número de divisões celulares ou picos entre amostras (Figuras 1 e 2). NOTA: Por exemplo, CFSE marcado, as células T específicas de OVA adoptivamente transferidos para ratinhos receptores que recebem o antigénio OVA vai sofrer proliferação activa em comparação com os ratinhos de controlo injectados com PBS 5,12. Além disso, é de salientar salientar que há muitas maneiras para analisar dados de proliferação de células CFSE, como demonstrado por Hawkins e seus colegas 14. </li></ol>

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The authors have nothing to disclose.

Neste protocolo, demonstramos um procedimento para a purificação de linfócitos de órgãos linfóides. purificação celular utilizando esférulas magnéticas triagem é um método rápido e simples que produz células alvo viáveis, altamente purificados. Passos críticos dentro do Protocolo A viabilidade celular e rendimento celular Mantendo a viabilidade das células de linhagem hematopoiéticas in vitro ...

Mature lymphocytes generally exist in the resting state if there is no pre-existing infection or inflammation in the individual. Therefore, it is important to retain the na&#239;ve status of lymphocytes during the isolation process before performing in vitro or in vivo functional assays. The key to ensuring consistent and reproducible results is to limit any unnecessary manipulation of the cells. 
Magnetic cell sorting utilizes antibodies and microbeads to label cells so as to enrich the cell population of interest. With this approach, there are two purification strategies: positive enrichment and negative depletion. Positive enrichment enriches the cell population of interest using an antibody that binds to the target cells. Negative depletion, on the other hand, depletes non-target cells, leaving the cell population of interest. In our lab, we prefer negative depletion to positive enrichment because the binding of antibodies to the target cells could potentially alter cell features and behavior. In fact, many established cell surface markers suitable for the isolation of a particular cell population are also functional receptors. 
Magnetic cell sorting not only yields highly pure populations of viable target cells, it is also less time-consuming and avoids the cellular stress induced by high-pressure flow used in fluorescence-activated cell sorting (FACS). By labeling the unwanted cell populations and depleting them using a magnetic separation column, we are able to perform rapid cell isolation without compromising the viability of the target cell population. In this protocol, we demonstrate the use of negative depletion strategies to purify na&#239;ve B cells or T cells.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>RPMI 1640 (without L-Glutamine)</td><td>Gibco</td><td>31870025</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td></td><td></td><td>Heat inactivated&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030024</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140114</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td></td></tr><tr><td>Anti-CD43 magnetic microbeads</td><td>Miltenyi Biotec</td><td>130-049-801</td><td>Mix well prior use</td></tr><tr><td>Streptavidin microbeads</td><td>Miltenyi Biotec</td><td>130-048-101</td><td>Mix well prior use</td></tr><tr><td>Anti-Annexin V magnetic beads</td><td>Miltenyi Biotec</td><td>130-090-201</td><td>Mix well prior use</td></tr><tr><td>MACS LD&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-042-901</td><td></td></tr><tr><td>96-well U-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>650180</td><td></td></tr><tr><td>96-well F-bottom sterile culture plate</td><td>Greiner Bio-one</td><td>655180</td><td></td></tr><tr><td>100 &amp;mu;m cell strainer mesh</td><td></td><td></td><td>To sterilize using UV radiation prior use</td></tr><tr><td>0.2 &amp;mu;m&amp;nbsp; sterile disposable filter units</td><td>Nalgene</td><td>567-0020</td><td>Can be substituted with any sterile filter device</td></tr><tr><td>CellTrace Violet</td><td>Invitrogen</td><td>C34557</td><td>CTV for short; alternative to CFSE</td></tr><tr><td>CellTrace Yellow</td><td>Invitrogen</td><td>C34567</td><td>CTY for short; alternative to CFSE</td></tr><tr><td>CellTrace Far Red</td><td>Invitrogen</td><td>C34564</td><td>CTFR for short; alternative to CFSE</td></tr><tr><td>Cell Proliferation Dye eFluor 670</td><td>eBioscience</td><td>65-0840</td><td>CPD670 for short; alternative to CFSE</td></tr><tr><td>PKH26</td><td>Sigma Aldrich</td><td>PKH26GL</td><td>PKH26, alternative to CFSE</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dextrose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P5655</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S5136</td><td></td></tr><tr><td>Phenol Red</td><td>Sigma Aldrich</td><td>P0290</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma Aldrich</td><td>C7902</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P5405</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>S7653</td><td>Can use from other sources</td></tr><tr><td>Magnesium chloride hexahydrate</td><td>Sigma Aldrich</td><td>M2393</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma Aldrich</td><td>M2643</td><td></td></tr><tr><td>Ammonium chloride</td><td>Sigma Aldrich</td><td>A9434</td><td></td></tr><tr><td>Tris-base</td><td></td><td></td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma Aldrich&amp;nbsp;</td><td>D8418</td><td></td></tr><tr><td>(5-(and 6-) carboxyfluorescein diacetate succinimidyl ester (CFSE)</td><td>Molecular Probes</td><td>C-1157</td><td>Reconstitute in DMSO</td></tr><tr><td>Phorbol 12,13-dibutyrate (PBDU, Phorbol ester)</td><td>Sigma Aldrich</td><td>P1269</td><td></td></tr><tr><td>A23187 (Calcium ionophore)</td><td>Sigma Aldrich</td><td>C7522</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies and recombinant protein&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CD11b biotin (clone m1/70)</td><td>Biolegend</td><td>101204</td><td>T cell depletion cocktail</td></tr><tr><td>CD11c biotin (clone N418)</td><td>Biolegend</td><td>117304</td><td>T cell depletion cocktail</td></tr><tr><td>Gr-1 biotin (clone RB6-8C5)</td><td>Biolegend</td><td>108404</td><td>T cell depletion cocktail</td></tr><tr><td>Ter119 biotin (clone Ter119)</td><td>Biolegend</td><td>116204</td><td>T cell depletion cocktail</td></tr><tr><td>TCR-&amp;gamma;&amp;delta; biotin (clone GL-3)</td><td>Biolegend</td><td>118103</td><td>T cell depletion cocktail</td></tr><tr><td>CD19 biotin (clone 6D5)</td><td>Biolegend</td><td>115504</td><td>T cell depletion cocktail</td></tr><tr><td>B220 biotin (clone RA3-6B2)</td><td>Biolegend</td><td>103204</td><td>T cell depletion cocktail</td></tr><tr><td>CD49b biotin (clone DX5)</td><td>Biolegend</td><td>108904</td><td>T cell depletion cocktail</td></tr><tr><td>CD4 biotin (clone GK1.5)</td><td>Biolegend</td><td>100404</td><td>T cell depletion cocktail</td></tr><tr><td>CD8 biotin (clone 53-6.7)</td><td>Biolegend</td><td>100704</td><td>T cell depletion cocktail</td></tr><tr><td>F(ab&amp;rsquo;)2 goat anti-mouse IgM (plate coated)</td><td>Jackson ImmunoResearch&amp;nbsp;</td><td>115-006-075</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-mouse CD40 mAb (plate coated)</td><td>Pharmingen&amp;nbsp;</td><td>553722</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-4</td><td>ProSpec&amp;nbsp;</td><td>Cyt-282</td><td></td></tr><tr><td>LPS from E. coli Serotype 055:B5</td><td>Sigma Aldrich</td><td>L-4005</td><td></td></tr><tr><td>Anti-CD3 (clone clone OKT3) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0037-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Anti-CD28 (clone clone 37.51) (plate coated)</td><td>eBioscience&amp;nbsp;</td><td>16-0281-85</td><td>50 &amp;micro;l/well for coating (96-well)</td></tr><tr><td>Recombinant IL-2</td><td>ProSpec</td><td>Cyt-370</td><td></td></tr><tr><td>Albumin from chicken egg white, Ovalbumin</td><td>Sigma Aldrich</td><td>A7641</td><td></td></tr></tbody></table>

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.

purificação magnética de células de linfócitos permite que os utilizadores para purificar uma população de células alvo num período relativamente curto de tempo. Utilizando o nosso protocolo de esgotamento, que foram capazes de aumentar a percentagem de células T CD8 (OT-I em recombinação-activação do gene-1 (RAG-1) -deficient ratinhos) a partir de 72,8% (antes da purificação) a 94,2% (após purificação; Figura 1A) 4,5. Estes linfócitos puri...

Watch this Scientific Journal Video about Isolation and Activation of Murine Lymphocytes at JoVE.com

Isolation and Activation of Murine Lymphocytes

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 ...

isolation-and-activation-of-murine-lymphocytes

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

21.0K visualizações.  Nanyang Technological University. 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.

Vídeo: Isolation and Activation of Murine Lymphocytes

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modelo murino de CD40-ativação de células B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Imunologia e Infecção

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 ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Protocolo de isolamento de células dendríticas derivadas de monócitos de rato e sua ativação subsequente em Vitro com Tumor de imunocomplexos

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Pesquisa do câncer

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolamento de células T CD4 + a partir dos nós linfáticos rato Usando Miltenyi Purificação MACS

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Biologia

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, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Isolamento, identificação e purificação de células epiteliais do timo murino

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolamento de Murino Linfonodo células estromais

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

An Efficient and High Yield Method for Isolation of Mouse Dendritic Cell Subsets

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen presenting molecules to initiate T-cell-mediated immunity. Dendritic cells can be separated into several phenotypically and functionally heterogeneous subsets. Three important subsets of splenic dendritic cells are plasmacytoid, CD8&#945;Pos and CD8&#945;Neg cells. The plasmacytoid DCs are natural producers of type I interferon and are important for anti-viral T cell immunity. The CD8&#945;Neg DC subset is specialized for MHC class II antigen presentation and is centrally involved in priming CD4 T cells. The CD8&#945;Pos DCs are primarily responsible for cross-presentation of exogenous antigens and CD8 T cell priming. The CD8&#945;Pos DCs have been demonstrated to be most efficient at the presentation of glycolipid antigens by CD1d molecules to a specialized T cell population known as invariant natural killer T (iNKT) cells. Administration of Flt-3 ligand increases the frequency of migration of dendritic cell progenitors from bone marrow, ultimately resulting in expansion of dendritic cells in peripheral lymphoid organs in murine models. We have adapted this model to purify large numbers of functional dendritic cells for use in cell transfer experiments to compare in vivo proficiency of different DC subsets.

Distinct dendritic cell subsets exist as rare populations in lymphoid organs, and therefore are challenging to isolate in sufficient numbers and purity for immunological experiments. Here we describe a high efficiency, high yield method for isolation of all of the currently known major subsets of mouse splenic dendritic cells.

Um método de rendimento eficiente e alta para o isolamento de subpopulações de células dendríticas Rato

Dendritic cells (DCs) are professional antigen-presenting cells primarily responsible for acquiring, processing and presenting antigens on antigen ...

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 ...

Isolando células endoteliais de capilares linfáticos dérmicos murinos

Murine Dermal Lymphatic Endothelial Cell Isolation

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic endothelial cells is an important process for lymphatic research, as it allows the performance of in vitro and biochemical experiments on the isolated cells. Moreover, the development of Cre-lox technology has enabled the tissue-specific deficiency of genes that cannot be globally targeted, leading to the precise determination of their role in the studied tissues. The dissection of the role of certain genes in lymphatic physiology and pathophysiology requires the use of lymphatic-specific promoters, and thus, the experimental verification of the expression levels of the targeted genes.
Methods for efficient isolation of lymphatic endothelial cells from wild-type or transgenic mice enable the use of ex vivo and in vitro assays to study the mechanisms regulating the lymphatic functions and the identification of the expression levels of the studied proteins. We have developed, standardized and present a protocol for the efficient isolation of murine dermal lymphatic endothelial cells (DLECs) via magnetic bead purification based on LYVE-1 expression. The protocol outlined aims to equip researchers with a tool to further understand and elucidate important players of lymphatic endothelial cell functions, especially in facilities where fluorescence-activated cell sorting equipment is not available.

Autor em destaque: Isolamento baseado em esferas magnéticas de células endoteliais linfáticas dérmicas murinas

This paper describes a method for magnetic bead-based isolation of murine endothelial cells from dermal lymphatic capillaries. The isolated lymphatic endothelial cells can be used for downstream in vitro experiments and protein expression analysis.

Isolamento de células endoteliais linfáticas dérmicas murinas

The lymphatic system participates in the regulation of immune surveillance, lipid absorption, and tissue fluid balance. The isolation of murine lymphatic ...