Pedimos gentilmente agradecer ao Dr. Clemens Wendtner para fornecer tmuCD40-Ligand-linha celular HeLa.

O protocolo para a geração de células CD40 de murino ativado B (mCD40B) de esplenócitos é dividido em duas partes: Parte A demonstra a preparação do ligante CD40 de murino (CD40L) expressando células HeLa (tmuCD40L HeLa), que será utilizado como placa obrigado células alimentadoras. Parte B descreve a real cultura CD40-B murinos. A. Preparação de células alimentadoras (tmuCD40L HeLa) O HeLa tmuCD40L é uma linha de células epiteliais humanas aderente, que nunca deve tornar-se completamente confluentes. As células são, portanto, dividido por duas vezes por semana. Cultura há mais de seis semanas não é recomendado. <ol><li> Remova o meio de idade a partir da cultura primária com uma pipeta estéril e lavar as células com 10 mL de PBS 1x. Aspirar o PBS após a lavagem. </li><li> Adicionar 4 mL de 1x Tripsina / EDTA em um frasco de 75 cm 2 por 10-15 minutos a 37 ° C. Use tocando suave para retirar as células. </li><li> Adicionar 10 mL de meio de tipo selvagem e giratória suavemente para parar a digestão com tripsina. </li><li> Transferir a suspensão de células em um tubo de 50 mL com uma pipeta estéril e girar a células para baixo a 265 xg por 7 minutos. </li><li> Remover o sobrenadante e ressuspender o pellet celular em 10 mL de meio de tipo selvagem. Conte o número de células de uma alíquota da suspensão de células e preparar três tubos de 50 mL com o número adequado de células: <ol><li> 2,5 x 10 6 células para subcultura </li><li> 0,4 x 10 6 células / poço de irradiação utilizadas para a cultura de células murinas CD40-B </li><li> restante para congelar (se necessário). </li></ol></li><li> Spin the células para baixo a 265 xg por 7 minutos. </li><li> Remover o sobrenadante. <ol><li> Para subcultura: ressuspender 2,5 x 10 6 células em 10 mL de médio seleção em um frasco de 75 centímetros de cultura de células 2 (densidade celular de 2,5 x 10 5 células / mL) e incubar as células a 37 ° C com 5% de CO 2. Dividir as células duas vezes por semana. </li><li> Para a cultura de células murinas CD40-B: Você precisa de 2,4 x 10 6 células para uma placa de 6 poços. Ressuspender as células HeLa em meio tmuCD40L tipo selvagem, a uma densidade de 0,2 x 10 6 células / mL e irradiar-los em 78 Gy. Placa de 2 mL da suspensão de células em cada poço e incube-os a 37 ° C com 5% de CO 2. Utilize este placas preparado para a estimulação de células B, quando as células HeLa são tmuCD40L aderente (pelo menos 4 horas: verificar a aderência do uso do microscópio, não espere mais de 24 h para iniciar a estimulação de células B). (Continue com B.) </li></ol></li></ol> B. murino CD40-cultura de células B I. Elaboração de esplenócitos para CD40-estimulação (dia 0): Por favor note: antes de continuar determinar que as células de alimentação são aderentes. Sempre adicionar novas soluções de murino imediatamente interleucina-4, β-mercaptoetanol e ciclosporina A para o meio de crescimento antes do uso. <ol><li> Dissecar baço de camundongos C57BL / 6 (haplótipo H-2b) e lavá-la duas vezes em 10-20 mL DMEM suplementado com Gentamicina [15μg/mL], transferindo-o com uma ponta de pipeta 10 ml de um tubo para outro. Evitar a transferência de contaminantes (por exemplo, o cabelo) do mouse. Mince baço por passagem através de um filtro de células (100μm) e enxaguar com outra peneira de 10 mL de meio. Spin down esplenócitos a 265 xg por 7 min em temperatura ambiente, ressuspender-los em 7ml muCD40-B médio de células, e os linfócitos separado por centrifugação Ficoll densidade. Para fazê-lo células da camada em 5 mL Mouse-Pancoll (pré-aquecido em temperatura ambiente) em um tubo de 15 ml e centrifugar a 450 xg por 30 min sem freio à temperatura ambiente. </li><li> Empate fora de a maioria da camada superior, deixando intacta a camada de linfócitos na interface. Transferência de camada de linfócitos a um tubo de 50 mL frescos, lave uma vez com 30mL DMEM com Gentamicina [15μg/mL] para baixo, girando a 265 xg por 7 min para remover outras células. Descartar o sobrenadante e ressuspender as células em 10 mL de mCD40-B médio de lavar roupa. Determinar o número de células em uma alíquota da suspensão de células. Determine o número de células por contagem uma alíquota da suspensão de células. </li><li> Spin para baixo a quantidade necessária de células em 265 xg por 7 minutos. Para uma placa de 6 poços 5 x 10 6 células / poço são necessários, portanto, 30 x 10 6 células por placa. </li><li> Remover o sobrenadante e ressuspender os linfócitos em 1,25 x 10 6 células / mL em mCD40-B meio de cultura fresco suplementado com 1 U / mL de IL-4 como fator de crescimento, 100 mM β-mercaptoetanol e 0,63 mcg / mL A ciclosporina para prevenir conseqüência de células-T (concentrações indicados referem-se a um meio de cultura mL!). </li><li> Retire o sobrenadante da placa de 6 poços de pré-incubados com células HeLa tmuCD40L. </li><li> Delicadamente adicionar 4 mL da suspensão de linfócitos (1,25 x 10 6 células / mL) a cada poço da placa de 6 poços. </li><li> Incubar a placa a 37 ° C com 5% de CO 2. </li&gt; <li> No dia 3 reculture as células (Continue com II.1.). </li></ol> II. Subcultura de células mCD40B (dia 3 e, em seguida, duas vezes por semana): <ol><li> Colheita aglomerado de células mCD40B por vigorosamente pipetando para cima e para baixo com uma pipeta de 10 mL e piscina-los em um tubo de 50 mL. </li><li> Girar em 265 xg por 7 minutos e substituir completamente o sobrenadante com mCD40-B médio de lavar roupa. Contando a quantidade de células de uma alíquota, gire a células para baixo a 265 xg por 7 minutos. Ressuspender as células em mCD40B mCD40-B meio de cultura a uma concentração de 0,75 x 10 6 células / mL. </li><li> Adicionar novas soluções de murino interleucina-4 em uma concentração de 1 U / mL, 100 Mercaptoethanol β-M e 0,63 mcg / mL ciclosporina A ao meio. </li><li> Remover o sobrenadante de uma placa de 6 poços de pré-incubados com células irradiadas tmuCD40L HeLa. </li><li> Delicadamente adicionar 4 mL da suspensão de células mCD40B (0,75 x 10 6 células / mL) a cada poço da placa de 6 poços. </li><li> Incubar as placas a 37 ° C com 5% de CO 2. </li><li> Novamente subcultura as células a cada 3-4 dias para acabar com alta pureza murino células CD40-B ativadas após um total de 14 dias. </li></ol> C. Resolução de problemas: O que se CD40-B células não crescem? <ol><li> Você tem verificado para CD40 ligante expressão de células de alimentação? </li><li> São a placa-bound células alimentadoras usado para a estimulação com mais de 24 horas? </li><li> Existe uma contaminação com micoplasma? </li><li> Foi a solução interleucina-4 utilizada para a suplementação recém descongeladas e que teve a atividade biológico adequado? </li><li> Foi β-mercaptoetanol e ciclosporina A adicionado na concentração correta? </li></ol><img src="/files/ftp_upload/1734/1734fig1a.jpg" alt="Figura 1-A" /> Figura 1a. <img src="/files/ftp_upload/1734/1734fig1b.jpg" alt="Figura 1 b" /> Figura 1b. <img src="/files/ftp_upload/1734/1734fig1d.jpg" alt="Figura 1 d" /> Figura 1d. <img src="/files/ftp_upload/1734/1734fig2.jpg" alt="Figura 2" /> Figura 2.

<ol>
	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100 (11), 2757-2765 (1997).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, e1464-e1464 (2008).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+generation+of+antigen-specific+cytotoxic+T+cells+using+retrovirally+transduced+CD40-activated+B+cells.">Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells.</a> J Immunol. 169 (4), 2164-2171 (2002).</li><li>Liebig, T. M., Fiedler, A., Zoghi, S., Shimabukuro-Vornhagen, A., Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+CD40-activated+B+cells.">Generation of human CD40-activated B cells.</a> J Vis Exp. 32, (2009).</li><li>von Bergwelt-Baildon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107 (7), 2786-2789 (2006).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63 (11), 2836-2843 (2003).</li><li>Ahmadi, T., Flies, A., Efebera, Y., Sherr, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+Ligand-activated,+antigen-specific+B+cells+are+comparable+to+mature+dendritic+cells+in+presenting+protein+antigens+and+major+histocompatibility+complex+class+I-+and+class+II-binding+peptides.">CD40 Ligand-activated, antigen-specific B cells are comparable to mature dendritic cells in presenting protein antigens and major histocompatibility complex class I- and class II-binding peptides.</a> Immunology. 124 (1), 129-140 (2008).</li><li>Bergwelt-Baildon, M. S. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99 (9), 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155 (2), 249-256 (2009).</li><li>Schultze, J. L., Grabbe, S., von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25 (12), 659-664 (2004).</li><li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+translocations+are+associated+with+poor+prognosis+in+chronic+lymphocytic+leukemia.">Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.</a> Blood. 107 (2), 742-751 (2006).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15 (13), 955-965 (2008).</li></ol>

Os experimentos foram realizados em conformidade com as diretrizes nacionais e europeus de manter laboratório animal. Referindo-se a lei alemã de bem-estar dos animais, os animais foram controlados regularmente pelas autoridades competentes.

Modificações deste protocolo são possíveis, especialmente quanto à origem do estímulo-CD40 e do tipo de estirpe do rato utilizado, rendendo também na geração eficiente de células CD40 de murino B ativadas. Ahmadi et al. têm mostrado resultados semelhantes para fibroblastos de rato transfectadas com murino CD40 ligante. Nesta apresentação contexto de xeno-antígeno pode ser excluída com certeza, mas estudos intensivos em matéria de segurança e toxicidade in vivo não deu sinais de inflamação ou auto-imunidade reação neste cenário com células transfectadas HeLa. No entanto, a melhor alternativa representa um murino solúvel CD40 ligante com a ativação e proliferação de atividade, ao mesmo tempo, que é a nosso conhecimento não disponíveis comercialmente. Este protocolo também funciona para linfócitos de ratos 129S2/SvPas (haplótipo H-2b), mas ainda não foi avaliada por linhagens de camundongos ainda mais até o momento. Além disso as diferenças de ajuste de densidade celular, a concentração de citocinas, a duração da ciclosporina A de tratamento, meios de cultura celular e pratos também podem levar a eficiente CD40-ativação de células B de camundongos com taxas de proliferação similar. No entanto, um intenso trabalho tem sido gasto na otimização deste sistema maximizando as taxas de ativação e proliferação, resultando no atual sistema.

A. Preparação de Media: <table border="1"><tbody><tr><td> Alimentador celular médio do tipo selvagem </td><td> Alimentador meio de seleção de célula </td><td></td></tr><tr><td> Reagente </td><td> Concentração </td><td> Reagente </td><td> Concentração </td></tr><tr><td> RPMI 1640 </td><td></td><td> RPMI 1640 </td><td></td></tr><tr><td> L-Glutamina </td><td> 300 mcg / mL </td><td> L-Glutamina </td><td> 300 mcg / mL </td></tr><tr><td> FBS </td><td> 10% </td><td> FBS </td><td> 10% </td></tr><tr><td> HEPES </td><td> 10 mM </td><td> HEPES </td><td> 10 mM </td></tr><tr><td> Gentamicina </td><td> 15 mcg / mL </td><td> Gentamicina </td><td> 15 mcg / mL </td></tr><tr><td></td><td></td><td> Higromicina B </td><td> 0,2 mg / mL </td></tr></table><table border="1"><tbody><tr><td> mCD40-B médio de lavar </td><td> mCD40-B meio de cultura </td></tr><tr><td> Reagente </td><td> Concentração </td><td> Reagente </td><td> Concentração </td></tr><tr></tr><tr><td> D-MEM </td><td> </td><td> D-MEM </td><td> </td></tr><tr><td> L-Glutamina </td><td> 580 mcg / mL </td><td> L-Glutamina </td><td> 580 mcg / mL </td></tr><tr><td> Glicose </td><td> 4,5 mg / mL </td><td> Glicose </td><td> 4,5 mg / mL </td></tr><tr><td> HEPES </td><td> 10 mM </td><td> HEPES </td><td> 10 mM </td></tr><tr><td> Gentamicina </td><td> 15 mcg / mL </td><td> Gentamicina </td><td> 15 mcg / mL </td></tr><tr><td> </td><td> </td><td> MEM </td><td> 0,1 mM (1x) </td></tr><tr><td> </td><td> </td><td> FBS </td><td> 10% </td></tr></tbody></table> B. Diversos Reagentes: <table border="1"><tr><td> Reagente </td><td> Companhia </td><td> Despacho n. </td></tr><tr><td> RPMI 1640 </td><td> Invitrogen Gibco </td><td> REF 21875 </td></tr><tr><td> D-MEM </td><td> Invitrogen Gibco </td><td> REF 41965 </td></tr><tr><td> Dulbeccos PBS (10x) </td><td> PAA </td><td> Cat Não H15-011 </td></tr><tr><td> Gencin </td><td> Selecione Delta </td><td> Arte Sem 7395800 </td></tr><tr><td> FBS </td><td> LONZA </td><td> Cat Não DE14-802C </td></tr><tr><td> Tampão HEPES </td><td> PAA </td><td> Cat Não S11-001 </td></tr><tr><td> MEM NEAA </td><td> Invitrogen Gibco </td><td> REF 11140 </td></tr><tr><td> Higromicina B </td><td> PAA </td><td> Cat Não P02-015 </td></tr><tr><td> Recombinante murino Interleucina-4 </td><td> Immunotools </td><td> Cat Não 12340045 </td></tr><tr><td> Ciclosporina A </td><td> Novartis </td><td> Cat Não NDC 0078-0109-01 </td></tr><tr><td> Tripsina / EDTA (10x) </td><td> Invitrogen Gibco </td><td> REF 15400-054 </td></tr></table> Fontes C. Diversos: <table border="1"><tbody><tr><td> Reagente </td><td> Companhia </td><td> Despacho n. </td></tr><tr><td> Pipeta estéril dicas </td><td> Sarstedt </td><td></td></tr><tr><td> 6-bem placa </td><td> NUNC </td><td> Não Cat 140675 </td></tr><tr><td> 50mL tubo cônico </td><td> BD Falcon ™ </td><td> Não Cat 352070 </td></tr><tr><td> Frasco de cultura de tecidos </td><td> Sarstedt </td><td> Cat Não 83.1813.002 </td></tr></tbody></table>

murine model of cd40-activation of b cells

A investigação sobre células B CD40 mostrou que melhora a sua capacidade de ativação apresentação de antígenos. Quando estimuladas com interleucina-4 e CD40 ligante (CD40L), células B humano pode ser expandido sem dificuldades a partir de pequenas quantidades de sangue periférico dentro de 14 dias para grandes quantidades de altamente pura CD40 células-B (&gt; 10<sup&gt; 9</sup&gt; Células por paciente) de doadores saudáveis, bem como pacientes com câncer<sup&gt; 04/01</sup&gt;. Células CD40-B expressam moléculas linfáticos importante nó de homing e podem atrair células T<em&gt; In vitro</em<sup&gt; 5</sup&gt;. Além disso, eles ocupam de forma eficiente, processar e apresentar antígenos às células T<sup&gt; 6,7</sup&gt;. Células CD40-B se mostraram não só ingênua prime, mas também expandir células T de memória<sup&gt; 8,9</sup&gt;. Portanto CD40-ativação de células B (CD40-células B) têm sido estudadas como uma fonte alternativa de imuno-estimulantes células apresentadoras de antígenos (APC) para a célula-base immunotherapy1, 5,10. A fim de estudar ainda mais se as células CD40-B induzir eficazes respostas de células T in vivo e estudar o mecanismo subjacente foi estabelecido um sistema de cultura celular para a geração de células CD40 de murino B ativadas. Usando esplenócitos ou purificada de células B de camundongos C57BL / 6 para CD40-ativação, as condições ideais foram identificados da seguinte forma: A partir de esplenócitos de camundongos C57BL / 6 (haplótipo H-2b) linfócitos são purificados por gradiente de densidade centrifugação e co-cultivados com HeLa células que expressam recombinante ligante CD40 de murino (tmuCD40L HeLa)<sup&gt; 11</sup&gt;. Células são recultured cada 3-4 dias e componentes-chave, como CD40L, a interleucina-4, Mercaptoetanol e ciclosporina A são reabastecidos. Neste protocolo que demonstrar como obter totalmente ativado murino CD40-B células (mCD40B) com semelhante APC-fenótipo CD40 humano para células-B (Fig. 1a, b). CD40-estimulação leva a um resultado rápido e expansão de alta pureza (&gt; 90%) CD19 + células B no prazo de 14 dias de cultura de células (Fig 1c, d). Para evitar a contaminação com os não-transfectadas células, a expressão do ligante CD40 de murino na transfectants tem que ser controlado regularmente (Fig. 2). Murino células CD40-activated B pode ser usado para estudo de células B de ativação e diferenciação, bem como para investigar seu potencial para funcionar como APC in vitro e in vivo. Além disso, eles representam uma ferramenta promissora para o estabelecimento de vacinação terapêutica ou preventiva contra tumores e vai ajudar a responder questões relativas à segurança e imunogenicidade desta abordagem<sup&gt; 12</sup&gt;.

Watch this Scientific Journal Video about Murine Model of CD40-activation of B cells at JoVE.com

Murine Model of CD40-activation of B cells

Neste vídeo, demonstramos o procedimento de ativação CD40-e expansão de células B murino de esplenócitos de camundongos C57BL / 6, que pode ser usado como um modelo de células apresentadoras de antígenos (APC) para estudar a indução de imunidade.

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 ...

murine-model-of-cd40-activation-of-b-cells

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

12.6K visualizações.  University Hospital of Cologne. Neste vídeo, demonstramos o procedimento de ativação CD40-e expansão de células B murino de esplenócitos de camundongos C57BL / 6, que pode ser usado como um modelo de células apresentadoras de antígenos (APC) para estudar a indução de imunidade.

Vídeo: Murine Model of CD40-activation of B cells

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

Indução e Avaliação de recombinação mudança de classe em purificada Células B murino

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Imunologia e Infecção

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

Produção recombinante Retroviral e infecção de células B

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

Modelo murino de asma induzida alergénio

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Ensaio de placas para Norovirus Murine

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Detecção de True IgE-expressando Rato B células da linhagem

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Geração de IgG Humana Recombinante de Anticorpos Monoclonais As células seleccionadas a partir de B imortalizadas

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Um modelo murino de Grupo B<em&gt; Streptococcus</em&gt; Colonização Vaginal

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

Detecção e enriquecimento da Rare células B antígeno-específica para a análise do fenótipo e função

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Análise de cisalhamento induzida pelo fluxo de migração de murino Marginal Zone B células In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

In Vitro Differentiation Model of Human Normal Memory B Cells to Long-lived Plasma Cells

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or mucosa and ensure humoral immunity. Due to their low frequency and location, the study of PCs is difficult in human. We reported a B to PC in vitro differentiation model using selected combinations of cytokines and activation molecules that allow to reproduce the sequential cell differentiation occurring in vivo. In this in vitro model, memory B cells (MBCs) will differentiate into pre-plasmablasts (prePBs), plasmablasts (PBs), early PCs and finally, into long-lived PCs, with a phenotype close to their counterparts in healthy individuals.&#160;We also built an open access bioinformatics tools to analyze the most prominent information from GEP data related to PC differentiation. These resources can be used to study human B to PC differentiation and in the current study, we investigated the gene expression regulation of epigenetic factors during human B to PC differentiation.

Using multi-step culture systems, we report an in vitro B cell to plasma cell differentiation model.

No modelo de diferenciação Vitro de células de memória humana Normal B de plasmócitos de vida longa

Plasma cells (PCs) secrete large amounts of antibodies and develop from B cells that have been activated. PCs are rare cells located in the bone marrow or ...