DRW es apoyado por subvenciones del NIH AI089972 y AI113217, y por el de la mucosa Estudios de Inmunología del equipo, y tiene una concesión de carrera para los científicos médicos del Fondo Burroughs Wellcome.

NOTA: Todos los experimentos descritos aquí estaban de acuerdo con el Comité de Cuidado y Uso de Animales Institucional (IACUC) directrices y aprobado por el Hospital de Niños de Investigación Animal (ARCH), Boston, Mass. 1. Preparación de los reactivos <ol><li> 1,000x IL-4 Stock: Diluir IL-4 de citoquinas a 20 mg / ml en H 2 O o 0,1% de albúmina de suero bovino (BSA). Distribuir en 200 ml de alícuotas de 1,5 ml tubos de microcentrífuga y almacenar a -20 ° C. </li><li> 1.000 x anti-CD40: Obtener del fabricante a 1,0 mg / ml, se almacena a 4 ° C. </li><li> B medios de estimulación de células: Para 425 ml de medio RPMI-1640, añadir 75 ml de suero de ternera fetal recién descongelado (FCS), 10 ml de 1,0 M de tampón HEPES, 5 ml de L-glutamina Stock, 5 ml de penicilina: Foto de estreptomicina, 5 ml Esenciales Medio no esenciales Aminoácidos l (MEM-NEAA), y 3,5 mínimas de 2-mercaptoetanol. Mantenga los medios a 4 ° C durante un máximo de una semana. Añadir IL-4 (20 ng / ml) y anti-CD40 (1.0 mg / ml) inmediatamente antes de su usosólo para el volumen de los medios necesarios. </li><li> 2% FCS-FACS Buffer: Añadir 10 ml de FCS a 490 ml de PBS 1x. Mantenga a 4 ° C. </li><li> 2x solución de tripsina-EDTA: Diluir 5 ml de 10x solución de tripsina-EDTA de stock (5,0 g de tripsina, EDTA 2,0 g por litro) en 20 ml de PBS 1x. Tienda tripsina-EDTA en 2x alícuotas de trabajo a 4 ° C. Se deja calentar hasta la temperatura ambiente antes de su uso. </li><li> El suero fetal de ternero: Mantenga FCS descongelaron a 4 ° C durante un máximo de dos semanas. Colocar en hielo 15 min antes de que el procedimiento de tinción citoplásmica. </li><li> -Neutral formalina tamponada: Preparar una solución de formalina al 10% (3,7% de paraformaldehído). La formalina es tóxico y, por tanto, manejar bajo una campana de extracción de laboratorio mientras usa el EPP apropiado. Formalina tienda a temperatura ambiente en un armario de almacenamiento de químicos. </li><li> Metanol: tienda metanol absoluto (100%) a -20 ° C hasta que las células están listas para ser permeabilizadas. Tenga en cuenta que el metanol es inflamable y se debe almacenar en un congelador apropiado. </li></ol> 2.La purificación y activación de células B <ol><li> Desde un bazo de ratón recién cosechado-, preparar una suspensión de células individuales presionando suavemente el órgano a través de un filtro de células de 70 micras con el émbolo de una jeringa de 3 ml. En un tubo de centrífuga cónico de 15 ml, recoger las células en 10 ml de 1x PBS y centrifugar a 300 xg durante 5 min a 4 ° C. </li><li> Decantar el sobrenadante y resuspender el precipitado en 1 ml de glóbulos rojos tampón de lisis durante 5 min. Neutralizar con 13,5 ml de 1x PBS y centrifugar a 300 xg durante 5 min. </li><li> (Opcional) separar magnéticamente las células B utilizando anti-CD45R (B220) marcado con perlas, siguiendo el protocolo de purificación MACS de acuerdo con las instrucciones del fabricante. </li><li> Estimular las células B purificadas a 1,0 x 10 6 células / ml en medio de estimulación de células B calentado que contiene IL-4 (20 ng / ml) y anti-CD40 (1.0 g / ml) durante 5 días a 37 ° C. Empiece con 8 ml de cultivo divididos en 2 pocillos de una placa de cultivo de 6 pocillos (4 ml cada una). </li><li> Compruebe culture diariamente para mantener a 1,0 x 10 6 células / ml. Ajustar la concentración dividiendo en otros pozos y diluyendo la cultura con los medios de comunicación la estimulación de células B que contiene IL-4 fresca y anti-CD40. </li></ol> 3. La tripsinización, Fijación y permeabilización <ol><li> La tripsinización <ol><li> Obtener hasta 1,0 x 10 7 de las células cultivadas en un tubo cónico de 15 ml, se centrifuga a 300 xg durante 5 min, y decantar el sobrenadante. </li><li> Lavar con 10 ml de PBS para eliminar la proteína residual de los medios de recogida de células. Repita el paso de centrifugación y decantar el sobrenadante. </li><li> Resuspender el sedimento celular en el volumen residual después de la decantación (aproximadamente 100 l). </li><li> Añadir cuidadosamente 800 l de temperatura ambiente 0,1% (2x) tripsina-EDTA a las células. Dispensar lentamente en un ángulo de 45 ° hacia abajo el lado del tubo cónico. </li><li> Cerrar el tubo y mezclar suavemente por la inclinación del tubo de manera que el líquido corre a lo largo de la longitud del tubo de tiempo de 5-6s. A, precipitado blanco fibroso puede formar en la solución. Limite el tiempo de tratamiento con tripsina a menos de un minuto. </li><li> Interrumpir la reacción tripsinización con 1 ml de FCS fríos. Diluir el FCS inmediatamente por la adición de 10 ml de PBS frío. </li><li> Centrifugar a 300 xg durante 5 min a 4 ° C, a continuación, decantar el sobrenadante y volver los tubos en hielo. </li></ol></li><li> Fijación <ol><li> Resuspender las células en el volumen residual después de la decantación. Si es necesario, llevar el volumen hasta 100 l con PBS frío. </li><li> En una campana de humos, añadir 800 l de solución de formalina tamponada neutra al 10% a las células y pipetear arriba y abajo. El tubo se calentará tras la adición de formalina. Incubar a 37 ° C durante 10 min. </li></ol></li><li> Permeabilización <ol><li> Configurar un vórtex de sobremesa a una velocidad de agitación suave. </li><li> Dibuje 9 ml de frío (-20 ° C) de metanol en un 10 ml pipeta serológica. </li><li> Sostenga el tubo que contiene las células sobre el mezclador de vórtice y empezar suavemente shaking las células. </li><li> Añadir metanol frío para el tubo de manera que cae gota a gota sobre las células mientras que el tubo todavía está sacudiendo de modo que el metanol se mezcla continuamente. Después de los primeros 2 ml de metanol la velocidad de dispensación se puede aumentar a una corriente lenta. </li><li> Deje que el tubo de sentarse en hielo durante 30 minutos para completar la permeabilización. Alternativamente, las células pueden almacenarse a -20 ° C hasta por un mes. </li></ol></li></ol> 4. La fluorescencia de tinción para IgG1 e IgE Class Switching <ol><li> Muestra Centrifugar a 300 xg durante 5 minutos y decantar la solución de metanol. Un precipitado blanco puede sedimentar con las células. </li><li> Añadir 10 ml de la temperatura ambiente 1x PBS y centrifugar a 300 xg durante 5 min para lavar las células. Repetir una vez. Nota: Lavar el pellet con temperatura ambiente PBS disuelve precipitado adicional que puede formar después del paso 4.1. </li><li> Realizar un lavado adicional con 10 ml de 2% de tampón FCS-FACS. </li><li> Distribuir las muestras a un redondo de 96 pocillos placa inferiory centrifugar a 300 xg durante 5 min. Decantar el sobrenadante y poner la placa en el hielo. </li><li> Las manchas en 60 l de 2% de tampón FCS-FACS por pocillo con las siguientes diluciones de anticuerpos: Anti-IgM RPE / Cy7, 1: 200, Anti-IgG1 PE, 1: 600, Anti-IgE FITC, 1: 100, Anti- CD45R (B220) PerCP-Cy5.5, 1: 200 </li><li> Después de 5 min de la tinción, se lava la placa con 150 l de 2% de tampón FACS-FCS y se centrifuga durante 5 min a 300 x g. </li><li> Resuspender en 400 l de 2% de tampón FACS-FCS y pipeta a través de un filtro de células de 40 m de nylon en un tubo de FACS 5 ml. </li></ol> 5. Utilice la estrategia de apertura de puerta a seguir: <ol><li> Aislar CD45R (B220) células positivas de la población de linfocitos voladura situado en la / parcela SSC FSC (Figura 1). </li><li> De las células positivas B220, usar el diagrama de IgE y IgG1 para revelar las poblaciones correspondientes. </li></ol>

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The authors have no disclosures.

La estimulación de células B de ratón esplénica en cultivo con anti-CD40 y IL-4 simulará T helper de tipo 2 (T H 2) las interacciones, el cambio de clase alentador IgG1 e IgE 5. Las células B pueden ser activados para la RSE en el contexto de esplenocitos totales 12 o células B esplénicas purificadas 11. Como se ha indicado en el protocolo (etapa 2.3), el enriquecimiento de células B es opcional, y es a discreción del experimentador para determinar si sería benefici...

Durante inmunoglobulina de cadena pesada (IgH) recombinación de cambio de clase (CSR) en ratones y seres humanos, la IgH μ exones de la región constante (Cμ) se eliminan y sustituyen por uno de varios conjuntos de los exones de la región constante aguas abajo (C H s) (por ejemplo Cγ, Cε, y Cα), resultando en un cambio de la producción de IgM a la producción de otras clases de Ig (por ejemplo, IgG, IgE, o IgA). RSE se produce dentro de conmutación (S), que son regiones 1-10 kb secuencias localizadas 5 &#39;a cada conjunto de C H 1 s. La activación inducida por citidina deaminasa (AID) enzima inicia la RSE a través de la actividad de citidina deamination. La IgE es un mediador clave de la enfermedad alérgica 2 y una mayor comprensión de cómo se produce y regulada puede abrir las puertas a nuevos enfoques terapéuticos para la enfermedad atópica IgE. En los ratones y los seres humanos, IgE es el isotipo Ig más estrechamente regulado. IgE normalmente se detecta en niveles miles de times menor que otro IgH isotipos 3, pero puede ser muy elevado en estados de enfermedad 4. Sin embargo, la RSE a la IgE no se conoce bien. In vitro la activación de células B usando IL-4 en combinación con anti-CD40 o LPS, induce CSR tanto a IgG1 e IgE 5. Las células B activadas expresan el receptor de baja afinidad IgE FcεRII / CD23 2,6, que se une la IgE soluble secretada en la cultura después de la RSE. Por lo tanto, al analizar con citometría de flujo, las células B con receptor de IgE unida mancha similar a las células B que expresan endógenamente IgE 7. Aunque se sabe que el ratón B células expresan el receptor de baja afinidad IgG FcγRIIB1 (CD32) 8, en nuestra experiencia, no parece interferir con la medición del cambio de clase a IgG1. Sin embargo, en la medición de conmutación IgE después de la activación de las células B en la cultura, unida a la superficie específica IgE puede oscurecer el análisis. Con los métodos de tinción comunes, las células no expresan IgE tiñen positivamente para IgE. Esbozado aquí es una estrategia que se ha utilizado para llevar a cabo ensayos de RSE y detectar verdaderas células IgE-B que expresan de ratones 9-11. El tratamiento de células B activadas con tripsina, un reactivo de laboratorio común para digerir las proteínas, elimina unido a la superficie tanto citofílicos y la membrana IgE. Permeabilización posterior y tinción citoplasmática de IgE por lo tanto revela las verdaderas células productoras de IgE.

<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 inmunoglobulina de linfocitos de cadena pesada (IgH) recombinación de cambio de clase (CSR) es un proceso en el que expresa inicialmente IgM cambia a otros isotipos de IgH, tales como IgA, IgE e IgG. Medición de la IgH CSR in vitro es un método clave para el estudio de una serie de procesos biológicos que van desde la recombinación y reparación del ADN a los aspectos de la inmunología molecular y celular. Ensayo in vitro RSE implica la superficie de medición expresión Ig de citometría de flujo en células B activadas. Si bien la medición de IgA e IgG subclases es sencillo, la medición de IgE por este método es problemático debido a la IgE soluble unión a FcεRII / CD23 expresado en la superficie de las células B activadas. Aquí se describe un procedimiento único para la medición precisa de IgE productoras de células B de ratón que han sido objeto de la RSE en la cultura. El método se basa en la escisión de tripsina-mediada de complejos IgE-CD23 en la superficie celular, lo que permite la detección de células de linaje B productoras de IgE por CYtinción toplasmic. Este procedimiento ofrece una solución conveniente para el análisis de citometría de flujo de la RSE a la IgE.

Este procedimiento se ha aplicado con éxito para estudiar la RSE a la IgE en las células B de ratón. Para demostrar la eficacia en la medición de la RSE, estimulamos las células B esplénicas de ratón con anti-CD40 y IL-4 como se describió previamente 11. Después de cinco días de estimulación, se recogieron las células y se procesaron usando el protocolo descrito anteriormente y se tiñeron con B220 (CD45R) anticuerpos IgM marcado con fluorescencia, IgG1, IgE, y. Cerrado en los linfocitos de voladu...

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.

Detección de True IgE que expresan las células del linaje B de ratón

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

10.8K vistas.  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.

Video: 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-activación de las células B

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

Investigación

Inmunología e Infección

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.

Inducción y evaluación de la recombinación de cambio de clase en las células murinas purificada B

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.

Recombinante de producción retrovirales y la infección 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.

Análisis rápido de aberraciones cromosómicas en linfocitos B de ratón 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.

Generación de recombinante humano IgG anticuerpos monoclonales a partir de células inmortalizadas B Ordenada

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.

Detección Molecular rápida y diferenciación de virus gripales A y 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.

Detección y enriquecimiento de Rare células B específicas de antígeno para el análisis del fenotipo y la función

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 ...

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.

Identificación y aislamiento de Oligopotent y linaje comprometido progenitores mieloides de la médula ósea de ratón

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.

Detección de copia baja número DNA Viral integradora formada por In Vitro la infección Hepatitis B

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

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.

Análisis de cortante inducida por el flujo de migración de las células B de zona Marginal de Murine 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, ...