DRW é suportado pelo NIH bolsas AI089972 e AI113217, e pela mucosa Estudos Imunologia Team, e detém um Prémio Carreira para médicos cientistas do Fundo Wellcome Burroughs.

NOTA: Todas as experiências descritas aqui foram de acordo com as diretrizes Comitê Cuidados e Uso de Animais Institucional (IACUC) e aprovado pelo Animal Hospital Research para a Infância (ARCH), Boston, Massachusetts. 1. Preparação dos Reagentes <ol><li> 1.000x IL-4 Stock: Diluir citocina IL-4 a 20 ng / ml em H2O ou 0,1% de albumina de soro bovino (BSA). Distribuir em alíquotas de 200 ul a tubos de microcentrífuga de 1,5 ml e armazenar a -20 ° C. </li><li> 1.000 vezes anti-CD40: obter do fabricante a 1,0 mg / ml, armazenar a 4 ° C. </li><li> Meios estimulação das células B: Para 425 ml de meio RPMI-1640, adicionar 75 ml de descongelado de fresco de soro fetal de vitelo (FCS), 10 ml de tampão HEPES 1,0 M, 5 ml de caldo de L-glutamina, 5 mL de Penicilina: stock de Estreptomicina, 5 ml essenciais Médio não-essenciais Aminoácidos jul (MAM-NEAA), e 3,5 mínimo de 2-mercaptoetanol. Mantenha o material a 4 ° C por até uma semana. Adicionar IL-4 (20 ng / ml) e anti-CD40 (1,0 ug / ml) imediatamente antes da utilizaçãoapenas com o volume de material necessário. </li><li> 2% de FCS em FACS Buffer: Adicionam-se 10 ml de FCS e 490 mL de PBS 1x. Mantenha a 4 ° C. </li><li> 2x solução de tripsina-EDTA: Diluir 5 ml de 10x solução estoque de tripsina-EDTA (tripsina 5,0 g, 2,0 g de EDTA por litro) em 20 ml de PBS 1x. Loja de tripsina-EDTA em 2x alíquotas trabalhando a 4 ° C. Deixa-se aquecer até à temperatura ambiente antes do uso. </li><li> Soro fetal bovino: Manter FCS descongeladas a 4 ° C por até duas semanas. Colocar em gelo 15 min antes do processo de coloração citoplasmática. </li><li> Neutro-tamponada de formalina: Prepare uma solução de formol a 10% (3,7% de paraformaldeído). Formalin é tóxico e, portanto, lidar com ele sob uma fume hood laboratório enquanto usava EPI adequado. Formalina loja à temperatura ambiente em um armário de armazenamento de produtos químicos. </li><li> Metanol: loja metanol absoluto (100%) a -20 ° C até as células estão prontas para ser permeabilizadas. Note-se que o metanol é inflamável e deve ser armazenado em freezer apropriado. </li></ol> 2.Purificação e activação de células B <ol><li> A partir de um baço recém-colhida rato, preparar uma suspensão de células individuais, pressionando suavemente o órgão através de um filtro de células 70 um com o êmbolo de uma seringa de 3 ml. Num tubo de centrífuga de 15 mL cónico, recolher as células em 10 ml de PBS 1x e centrifugar a 300 xg durante 5 min a 4 ° C. </li><li> Decantar o sobrenadante e ressuspender o pellet em 1 ml de células vermelhas do sangue tampão de lise durante 5 min. Neutraliza-se com 13,5 ml de PBS 1x e centrifugar a 300 xg durante 5 min. </li><li> (Opcional) magneticamente separar as células B, utilizando anti-CD45R (B220) marcado com grânulos, seguindo o protocolo de purificação MACS acordo com as instruções do fabricante. </li><li> Estimular as células B purificadas a 1,0 x 10 6 células / ml em meios aquecida a estimulação das células B contendo IL-4 (20 ng / ml) e anti-CD40 (1,0 ug / ml) durante 5 dias a 37 ° C. Comece com 8 ml de cultura divididos em dois poços de uma placa de cultura de 6 poços (4 ml cada). </li><li> Verifique culture diária para manter a 1,0 x 10 6 células / ml. Ajustar a concentração através da divisão em outras cavidades e a diluição da cultura com meio de estimulação das células B fresco contendo IL-4 e anti-CD40. </li></ol> 3. A tripsinização, Fixação e permeabilização <ol><li> Tripsinizaï¿½o <ol><li> Recolhe-se a 1,0 x 10 7 de células de cultura num tubo de 15 ml, centrifugar a 300 xg durante 5 minutos, e decanta-se o sobrenadante. </li><li> Lava-se com 10 ml de PBS para remover a proteína residual a partir do suporte de recolha de células. Repetir o passo de centrifugação e decantar o sobrenadante. </li><li> Ressuspender o sedimento de células no volume residual após decantação (cerca de 100 uL). </li><li> Adiciona-se cuidadosamente 800 ul de 0,1% temperatura ambiente (2x) de tripsina-EDTA para as células. Dispensar lentamente num ângulo de 45 ° para o lado do tubo cónico. </li><li> Fechar o tubo e misturar suavemente pela inclinação do tubo de modo que o líquido corre ao longo do comprimento do tubo de tempo 5-6s. Um precipitado pegajoso, branco pode formar-se na solução. Limite o tempo de tripsinização para menos de um min. </li><li> Extingue-se a reacção tripsinização com 1 ml de FCS a frio. Dilui-se o FCS imediatamente por adição de 10 ml de PBS frio. </li><li> Centrifuga-se a 300 xg durante 5 min a 4 ° C, depois decanta-se o sobrenadante e devolver os tubos em gelo. </li></ol></li><li> Fixação <ol><li> Ressuspender as células do volume residual, após decantação. Se necessário, levar o volume até 100 ml com PBS frio. </li><li> Numa hotte, adicionar 800 ul de solução de formalina tamponada neutra a 10% para as células e pipetar para cima e para baixo. O tubo vai aquecer após a adição de formalina. Incubar a 37 ° C durante 10 min. </li></ol></li><li> Permeabilização <ol><li> Configurar um misturador de bancada vortex a uma velocidade agitação suave. </li><li> Desenhar 9 ml de frio (-20 ° C) de metanol em 10 ml de uma pipeta serológica. </li><li> Segurar o tubo contendo as células ao longo do misturador de vórtice e suavemente começar tremendog das células. </li><li> Adicionar metanol frio para o tubo assim que cai gota a gota sobre as células, enquanto o tubo está ainda agitação, de modo que o metanol é se mistura continuamente. Depois dos primeiros 2 ml de metanol a velocidade de distribuição pode ser aumentada para um caudal lento. </li><li> Deixe o tubo de sentar em gelo durante 30 min para completar a permeabilização. Alternativamente, as células podem ser armazenadas a -20 ° C durante até um mês. </li></ol></li></ol> 4. A fluorescência de coloração para IgG1 e IgE Classe Switching <ol><li> Amostra Centrifugar a 300 xg durante 5 minutos e decantar a solução de metanol. Um precipitado branco podem sedimentar com as células. </li><li> Adicionar 10 ml de 1x PBS temperatura ambiente e centrifugar a 300 xg durante 5 minutos para lavar as células. Repetir uma vez. Nota: A lavagem do pellet com temperatura ambiente PBS dissolve precipitado extra que podem formar após o passo 4.1. </li><li> Realizar uma lavagem adicional com 10 ml de 2% FCS-tampão de FACS. </li><li> Distribuir amostras de 96 poços rodada placa de fundoe centrifugar a 300 xg durante 5 min. Decantar o sobrenadante e colocar a placa no gelo. </li><li> Mancha em 60 ul de tampão de 2% de FCS em FACS por poço com as seguintes diluições de anticorpos: anti-IgM RPE / Cy7, 1: 200, anti-IgG1 de PE, 1: 600, FITC anti-IgE, 1: 100, Anti CD45R (B220), PerCP-Cy5.5, 1: 200 </li><li> Após 5 min de coloração, lavar a placa com 150 ul de 2% de FCS-tampão de FACS e centrifugar durante 5 minutos a 300 x g. </li><li> Ressuspender em 400 ul de tampão de 2% de FCS em FACS e por meio de uma pipeta de 40 um coador de células de nylon para um tubo de 5 ml de FACS. </li></ol> 5. Use a Estratégia Gating seguir: <ol><li> Isolar CD45R (B220) células positivas da população de linfócitos jateamento localizado no / parcela SSC FSC (Figura 1). </li><li> A partir das células positivas B220, utilizar o lote de IgE e IgG1 para revelar as populações correspondentes. </li></ol>

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The authors have no disclosures.

A estimulação de células B do baço de rato em cultura com anti-CD40 e IL-4 irá simular tipo T helper 2 (T H 2) interacções, troca de classe encorajador IgG1 e IgE 5. As células B podem ser ativados para a RSE no âmbito do total esplenocitos 12 ou células B do baço como purificados 11. Como foi observado no protocolo (passo 2.3), o enriquecimento de células B é opcional, e fica ao critério do experimentador para determinar se seria benéfico. Separação magnétic...

Durante cadeia pesada de imunoglobulina (IgH) recombinação mudança de classe (CSR) em ratos e seres humanos, o IgH μ exons região constante (Cμ) são eliminados e substituídos por um dos vários conjuntos de jusante exons região constante (C H s) (por exemplo, Cy, Cε, e Ca), resultando numa passagem da produção de IgM para a produção de outras classes de Ig (por exemplo, IgG, IgE, ou IgA). RSE ocorre dentro de regiões de troca (S), que são sequências de 1-10 kb localizado 5 &#39;em cada conjunto de C H 1 s. A enzima Activação Induzida citidina-desaminase (AID) inicia RSE através da actividade de citidina de desaminação. IgE é um mediador chave da doença alérgica 2 e uma maior compreensão de como IgE é produzida e regulamentada pode abrir as portas para novas abordagens terapêuticas para a doença atópica. Em ratos e seres humanos, IgE é o isotipo Ig mais rigidamente controlado. IgE é normalmente detectada em níveis milhares de times menos do que outros isotipos de IgH 3, mas pode ser muito elevado em estados de doença 4. No entanto, a RSE para IgE é completamente compreendida. In vitro a activação de células B com IL-4 em combinação com anti-CD40 ou LPS, induz RSE para ambos IgG1 e IgE 5. Células B ativadas expressar a baixa afinidade receptor IgE FceRII / CD23 2,6, que liga IgE solúvel secretada em cultura após CSR. Portanto, quando a análise por citometria de fluxo, as células B com receptor de IgE ligado a mancha de modo semelhante às células B expressando IgE 7 endogenamente. Embora se saiba que o mouse B células expressam a baixa afinidade IgG receptor FcγRIIB1 (CD32) 8, em nossa experiência, não parece interferir com a medição da classe de comutação para IgG1. No entanto, quando se mede a IgE de comutação após a activação das células B em cultura, não específica ligada à superfície IgE pode obscurecer a análise. Com métodos de coloração comuns, as células não-IgE-expressando manchar positivamente para IgE. Esboçado aqui é uma estratégia que tem sido utilizada para realizar ensaios de RSE e detectar células B expressando IgE verdadeiros de ratos 9-11. O tratamento de células B activadas com tripsina, um reagente de laboratório comum para digerir a proteína, remove tanto cytophylic superfície e ligada à membrana de IgE. Permeabilização e subsequente coloração citoplasmática para IgE revela, assim, as células produtoras de IgE verdadeiros.

<table border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:301px;">Name</td>
			<td style="width:179px;">Company</td>
			<td style="width:135px;">Catalog Number</td>
			<td style="width:343px;">Comments</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Mouse IgE FITC</td>
			<td>BD Pharmingen</td>
			<td>553415</td>
			<td>clone R35-72</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgG1 PE</td>
			<td>BD Pharmingen</td>
			<td>550083</td>
			<td>clone A85-1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Methanol</td>
			<td>BDH</td>
			<td>BDH1135-4LP</td>
			<td>Keep at -20 &#176;C</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 40 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Falcon Cell Strainer 70 &#181;m Nylon</td>
			<td>Corning</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Anti-Hu/Mo CD45R(B220) PerCP-Cy5.5</td>
			<td>eBioscience</td>
			<td>45-4052-82</td>
			<td>clone RA3-6B2</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">anti-Mouse/Rat CD40</td>
			<td>eBioscience</td>
			<td>16-0402-85</td>
			<td>clone HM40-3</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">RPMI Medium 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MEM-NEAA (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Phosphate Buffered Saline (10X)</td>
			<td>Lifetechnologies (Corning)</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="66">
			<td height="66" style="height:67px;">MACS CD45R (B220) microbeads&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-501</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">MACS purification column</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">IL-4</td>
			<td>PeproTech</td>
			<td>214-14</td>
			<td>Reconstitute in water or 0.1% BSA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Formalin Solution, Neutral Buffered, 10%</td>
			<td>Sigma Aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Trypsin-EDTA Solution (10X)</td>
			<td>Sigma Aldrich</td>
			<td>T4174-100mL</td>
			<td>5.0g Trypsin, 2.0g EDTA&#376;4Na per Liter of 0.9% NaCl</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Penicillin-Streptomycin</td>
			<td>Sigma Aldrich</td>
			<td>P0781-100mL</td>
			<td>10,000U Penicillin; 10 mg/mL Streptomycin (100X)</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">L-Glutamine 200mM</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">2-mercaptoethanol (99%)</td>
			<td>Sigma Aldrich</td>
			<td>M6250-100mL</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Red cell lysis buffer</td>
			<td>Sigma Aldrich</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">Rat Anti-Mouse IgM RPE/cy7</td>
			<td>SouthernBiotech</td>
			<td>1140-17</td>
			<td>clone 1B4B1</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;">HyClone Fetal Calf Serum</td>
			<td>Thermo</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="161">
			<td height="161" style="height:161px;">B cell Stimulation media</td>
			<td></td>
			<td></td>
			<td style="width:343px;">B cell stimulation media consists of RPMI with fetal calf serum (15%), 20 mM HEPES, 1X MEM-NEAA, 2.0mM L-glutamine,&#160; 1X Penicillin-Streptomycin (Penicillin:100 U/ml, Streptomycin: 100 &#181;g/ml), Beta-mercaptoethanol (7 &#181;L/L), IL-4 (20 ng/mL), and anti-CD40 (1 &#181;g/mL)</td>
		</tr>
	</tbody>
</table>

detection of true ige-expressing mouse b lineage cells

B imunoglobulina linfócitos cadeia pesada (HGI) recombinação mudança de classe (CSR) é um processo em que, inicialmente, expressa IgM muda para outros isotipos CMI, tais como IgA, IgE e IgG. Medição de IgH RSE in vitro é um método fundamental para o estudo de uma série de processos biológicos variam de recombinação e reparação do ADN de aspectos da imunologia molecular e celular. Ensaio in vitro RSE envolve a superfície de medição de citometria de fluxo a expressão de Ig em células B activadas. Embora a medição de IgA e IgG subclasses é simples, medição de IgE por este método é problemático devido à ligação de IgE a FceRII solúvel / CD23 expresso na superfície de células B activadas. Aqui, descrevemos um processo único para a medição precisa de IgE células B de ratinho produtora que tenham sido submetidos a RSE em cultura. O método baseia-se na clivagem mediada por tripsina de complexos IgE-CD23 na superfície das células, permitindo a detecção de células produtoras de IgE da linhagem B por cycoloração toplasmic. Este procedimento oferece uma solução conveniente para a análise de citometria de fluxo de CSR para IgE.

Este procedimento foi implementado com sucesso para estudar CSR para IgE em células B de ratinho. Para demonstrar a eficiência na medição RSE, que estimulou as células B do baço de ratinho com anticorpo anti-CD40 e IL-4 como descrito anteriormente 11. Depois de cinco dias de estimulação, as células foram recolhidas e processadas utilizando o protocolo descrito acima e coradas com fluorescência marcado IgM, IgG1, IgE, anticorpos e B220 (CD45R). Recolhidas em linfócitos de detonação (Figura...

Watch this Scientific Journal Video about Detection of True IgE-expressing Mouse B Lineage Cells at JoVE.com

Detection of True IgE-expressing Mouse B Lineage Cells

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 ...

detection-of-true-ige-expressing-mouse-b-lineage-cells

Research

JoVE Journal

Immunology and Infection

10.9K visualizações.  Brigham and Women's Hospital and Harvard Medical School. 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.

Vídeo: Detection of True IgE-expressing Mouse B Lineage Cells

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

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 ...

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 ...

Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency and type of chromosome aberrations in different cell types allows defects in DNA repair pathways to be elucidated. Understanding mammalian DNA repair biology has been greatly helped by the production of mice with knockouts in specific genes. The goal of this protocol is to quantify genomic instability in mouse B lymphocytes. Labeling of the telomeres using PNA-FISH probes (peptide nucleic acid - fluorescent in situ hybridization) facilitates the rapid analysis of genomic instability in metaphase chromosome spreads. B cells have specific advantages relative to fibroblasts, because they have normal ploidy and a higher mitotic index. Short-term culture of B cells therefore enables precise measurement of genomic instability in a primary cell population which is likely to have fewer secondary genetic mutations than what is typically found in transformed fibroblasts or patient cell lines.

DNA repair pathways are essential for maintenance of genomic integrity and preventing mutation and cancer. The goal of this protocol is to quantify genomic instability by direct observation of chromosome aberrations in metaphase spreads from mouse B cells using fluorescent in situ hybridization (FISH) for telomeric DNA repeats.

Rápida análise de aberrações cromossômicas em linfócitos B mouse por PNA-FISH

Defective DNA repair leads to increased genomic instability, which is the root cause of mutations that lead to tumorigenesis. Analysis of the frequency ...

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, ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Rápida Detecção Molecular e diferenciação de Influenza vírus A e B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

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 ...

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Deteção de ativação de Inflammasome e morte celular de Pyroptotic nos macrófagos da medula óssea-derivado murino

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

Identificação e isolamento de oligopotentes e confirmada por linhagem mieloides progenitores da medula óssea de rato

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

Deteção de cópia baixa número de DNA Viral integrado formado por In Vitro infecção de hepatite B

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...