Este trabajo es apoyado por el NIH subvención R00 CA160574 (SFB) y por una beca del Programa de Entrenamiento de Biotecnología Rutgers (a SMM).

Este procedimiento fue aprobado por el Comité de Cuidado y Uso de Animales institucional en Rutgers, la Universidad Estatal de Nueva Jersey. Los ratones fueron tratados de acuerdo con la Guía del NIH para el Cuidado y Uso de Animales de Laboratorio. Los científicos deberían consultar a sus organizaciones animalistas nacionales e institucionales de las normas establecidas y aprobadas. Antes de comenzar, preparar las soluciones enumeradas en la Tabla 1. Aislamiento Celular 1 B y Activación La eutanasia a un ratón utilizando CO 2 seguido por dislocación cervical. Coloque el ratón para que el lado izquierdo del cuerpo está en marcha y rociar el ratón con etanol al 70% hasta que la piel está húmeda. Diseccionar el bazo y el lugar en un 35 mm placa de cultivo de tejidos con tampón de lavado 2 ml. En una campana de flujo laminar, romper suavemente el bazo en la placa de cultivo de tejidos utilizando el extremo plano de una jeringa de 5 ml. <ol><li> Inserte una malla de nylon de 70 micras en un 50tubo ml y transferir la muestra de bazo en 2 ml de tampón de lavado al interior de la malla de nylon. Interrumpir suavemente el bazo en la malla utilizando el extremo plano de la jeringa. </li><li> Enjuague la parte posterior de la jeringa y la placa de 35 mm con 8 ml de tampón de lavado y filtrado a través de la malla de nylon 70 micras. Centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Aspirar y desechar el sobrenadante y resuspender el precipitado en 3 ml de tampón de lisis ACK. Pipetear hacia arriba y abajo para interrumpir brevemente montones e incubar durante 5 min. Añadir 10 ml de tampón de lavado y se centrifuga a 300 g durante 10 min a 4 ° C. </li><li> Aspirar y desechar el sobrenadante y resuspender el precipitado tocando el fondo del tubo y luego con la pipeta en 1 ml de tampón de lavado. Añadir 50 l de anti-CD43 MACS micro-cuentas e incubar en hielo durante 30 min. Añadir 10 ml de tampón de lavado, mezclar suavemente y centrifugar a 300 xg durante 10 min a 4 ° C. Añadir 10 ml de tampón de lavado, mezclar suavemente y centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Establecer unColumna Mini-MACS mediante la colocación de una columna en el imán MACS. Coloque un tubo cónico de 15 ml etiquetados bajo la columna. Añadir 1 ml de tampón de lavado a la columna y dejar correr a través de al tubo de 15 ml. </li><li> Aspirar el sobrenadante y resuspender en 1 ml de tampón de lavado. Cargar la muestra en la columna después el tampón de lavado se ha ejecutado a través. </li><li> Después de que la muestra ha ejecutar a través de la columna, añadir 1 ml de tampón de lavado para lavar la columna. Añadir 7 ml de tampón de lavado directamente a la muestra recogida en el tubo de 15 ml. </li><li> Contar las células utilizando un hemocitómetro. Transferir el volumen requerido de células necesarias para 5.000.000 células / pocillo en un tubo de 50 ml. Centrifugar a 300 xg durante 10 min a 4 ° C. Suplemento medio de células B con LPS 1: 500 y 1 IL4: 1000. </li><li> Aspirar el sobrenadante y añadir el volumen apropiado de medio de células B suplementado necesaria para la concentración final de 1.000.000 células / ml. </li><li> Células de la placa en placas de 6 pocillos con 5 ml de células a 1.000.000 de células / ml y colocar en un humidified incubadora de cultivo celular a 37 ° C, 5% de CO 2. </li></ol> Fijación Cell 2 B <ol><li> Añadir 50 l de colcemid al pozo que contiene 5 ml de las células B (para una concentración final de 100 ng / ml) y se incuba 1 hora a 37 ° C. Precalentar 75 mM KCl solución en un baño a 37 ° C. Transferencia de las células B a un tubo de 15 ml etiquetados. Cubra la etiqueta con cinta y centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Aspirar el sobrenadante dejando 1 ml en el tubo y resuspender el pellet mediante pipeteo. Añadir 5 ml de la caída de KCl precalentado a gota mientras golpea o pulsante en un vórtice. </li><li> Añadir 10 ml de KCl y mezclar invirtiendo. Incubar en un 37 ° C baño de agua durante 15 min. Añadir 5 gotas de fijador fresco y mezcla por inversión. Centrifugar a 300 xg durante 10 min a 4 ° C. Aspirar el sobrenadante dejando 1 ml en el tubo y resuspender el pellet pipeteando arriba y abajo. </li><li> Añadir 5 ml de fijador fresco gota a gota mientras golpea o pulsante en un vortex. Añadir otros 10 ml de fijador y se incuba durante 30 min a temperatura ambiente. </li><li> Centrifugar a 300 xg durante 10 min a 4 ° C. Aspirar el sobrenadante dejando 1 ml en tubo y resuspender con la pipeta. Añadir 14 ml de fijador fresco y centrifugar a 300 xg durante 10 min a 4 ° C. </li><li> Repita el paso 2.7 en dos ocasiones más. </li><li> Añadir 14 ml de fijador. Selle los tapones con Parafilm y almacenar toda la noche a -20 ° C. </li></ol> 3 Preparación de las extensiones de cromosomas en metafase <ol><li> Establezca una cámara ambiental regulada a 22,9 ° C y 52% de humedad. </li><li> Centrifugar las células B fijado a 300 xg durante 10 min a 4 ° C. Aspirar fijador dejando 70 l en el tubo y resuspender mediante pipeteo. </li><li> Coloque las diapositivas marcadas en la cámara de humedad en un ángulo de 35 °. Gota 35 l de las células B resuspendidas en un portaobjetos y soltar dos portaobjetos por muestra. Deje que los portaobjetos se sequen en la cámara de humedad durante 30 minutos y luego guardar las diapositivasen una caja de portaobjetos a 37 ° C. </li></ol> 4. telómeros PNA FISH <ol><li> Preparación de la sonda <ol><li> Pre-caliente formamida desionizada a 37 ° C. Mezclar 2 l de sonda de APN de los telómeros con 7 l de formamida desionizada precalentada y se incuba a 37 ° C con agitación durante 1 hr. </li><li> Pre-caliente mezcla maestra FISH a 37 ° C. Añadir 7 l precalentado mezcla maestra FISH a la mezcla del paso 4.1.1 y se incuba a 37 ° C durante 1-3 horas. </li><li> Desnaturalizar la mezcla de la sonda durante 8 minutos a 80 ° C. Pre-hibridar la sonda durante 1 hora a 37 ° C. </li></ol></li><li> Pretratamiento de portaobjetos cromosómicos <ol><li> Pre-calentar un tarro de cristal deslizable tinción que contiene 50 ml de HCl 0,01 M a 37 ° C en un baño de agua. </li><li> Colocar los portaobjetos en un tarro de cristal deslizable tinción diferente que contiene 2x SSC durante 5 min a temperatura ambiente. </li><li> Añadir 2 l de pepsina de stock al vidrio precalentado slide-tinción frasco y mezclar invirtiendo. Transferir las diapositivas a este sl vidriojar ide-tinción e incubar 90 segundos a 37 ° C. </li><li> Lave las diapositivas 2x en un frasco de vidrio deslizable tinción diferente que contiene 1x PBS durante 5 minutos cada uno a temperatura ambiente, agitando. A continuación, lavar los portaobjetos durante 5 minutos en 1x PBS / MgCl2 a temperatura ambiente, agitando. </li><li> Transferir los portaobjetos a un tarro de cristal deslizante de tinción recién preparada que contenía 1% de formaldehído-/ 1x PBS / MgCl $ 2 durante 10 min a temperatura ambiente. </li><li> Lavar los portaobjetos durante 5 minutos en un frasco de vidrio deslizable tinción diferente que contiene 1x PBS a temperatura ambiente, agitando. </li><li> Preparar una serie de deshidratación de etanol por tener vidrio separada frascos de deslizamiento de tinción que contienen 70%, 90% y 100% de etanol. </li><li> Transferir los portaobjetos a cada frasco durante 3 min, empezando con etanol al 70%, luego 90% de etanol, y luego etanol al 100%. Coloque los tarros de etanol a -20 ° C cuando haya terminado. </li><li> Permitir las diapositivas se sequen al aire tocando el exceso de etanol en una toalla de papel y apuntalar las diapositivas en un ángulo de 35 °. Una vez seco, mark un área de 18 x 18 mm para la hibridación en la parte posterior de las diapositivas utilizando una pluma de diamante. </li></ol></li><li> La desnaturalización de las diapositivas para FISH <ol><li> Aplicar formamida 120 l 70% desionizada / 2X SSC a un cubreobjetos de 24 x 60 mm. Toca la diapositiva a la cubreobjetos. Desnaturalizar la diapositiva a 80 ° C en una placa caliente durante 90 segundos. </li><li> Rápida y cuidadosamente deslice el cubreobjetos y colocar el portaobjetos en hielo frío etanol al 70% durante 3 min, seguido de etanol 90% y etanol al 100% durante 3 min cada uno. Permitir las diapositivas se sequen al aire. </li><li> Preparar una cámara húmeda por el forro una caja de plástico con tapa hermética y un pañuelo de papel mojado. El papel debe estar húmedo, pero no saturado con agua. </li><li> En una cámara húmeda, aplique la mezcla de sondas pre-recocido desde el paso 4.1.3 a la zona marcada en el portaobjetos, cubra con una tapa deslizante mm 18 x 18 y se incuba a 37 ° C durante 1 hora. </li></ol></li><li> Los lavados post-incubación <ol><li> Pre-caliente 50% de formamida / SSC 2x, 1x SSC, y 4x SSC / 0,1% de Tween-20 en unos 45 °; C baño de agua. </li><li> Retire con cuidado el cubreobjetos y lavar los portaobjetos en pre-calentado 50% formamida / 2 × SSC 3xfor 5 minutos cada uno en la oscuridad y temblando. Se lavan los portas en pre-calentado 1x SSC 3xfor 5 minutos cada uno, temblando. Finalmente, lavar los portaobjetos en pre-calentado 4x SSC / 0,1% de Tween-20 3xfor 5 min cada uno. </li><li> Teñir las diapositivas durante 3 min en DAPI en una luz protegida vidrio deslizable tinción frasco. </li><li> Lavar los portaobjetos durante 5 minutos en 2x SSC, temblando. Aplique 35 l de medio de montaje Mowiol, cubrir con 24 x 60 mm cubreobjetos y almacenar las diapositivas a 4 ° C. </li></ol></li></ol> 5. microscópica Análisis de Cromosomas <ol><li> Analizar las diapositivas en metafase utilizando un microscopio de epi-fluorescencia estándar. Utilice una plataforma de imágenes con una etapa automatizada como la plataforma MetaSystems Metafer para obtener mejores resultados. Para cada extensión metafase, recoger una imagen de fluorescencia DAPI para visualizar los cromosomas, y una imagen de la señal fluorescente del telómero pbata. (Por ejemplo, Cy3 otros compuestos fluorescentes están disponibles y funcionan igual de bien.) </li><li> Superposición de las imágenes con un programa de análisis de imagen y analizar. </li></ol>

<ol>
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N., Reijo Pera, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metaphase+spreads+and+spectral+karyotyping+of+human+embryonic+stem+cells.">Metaphase spreads and spectral karyotyping of human embryonic stem cells.</a> Cold Spring Harb Protoc. 2008, (2008).</li><li>Schreck, R. R., Disteche, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+4.">Chapter 4.</a> Chromosome banding techniques. Unit 2, (2001).</li><li>Gilley, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-PKcs+is+critical+for+telomere+capping.">DNA-PKcs is critical for telomere capping.</a> Proc Natl Acad Sci USA. 98 (26), 15084-15088 (2001).</li><li>Bolzan, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+aberrations+involving+telomeres+and+interstitial+telomeric+sequences.">Chromosomal aberrations involving telomeres and interstitial telomeric sequences.</a> Mutagenesis. 27 (1), 1-15 (2012).</li><li>Bolzan, A. D., Telomeres Bianchi, 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=interstitial+telomeric+repeat+sequences+and+chromosomal+aberrations.">interstitial telomeric repeat sequences and chromosomal aberrations.</a> Mutat Res. 612 (3), 189-214 (2006).</li><li>Poon, S. S., Lansdorp, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+18.">Chapter 18.</a> Quantitative fluorescence in situ hybridization (Q-FISH). Unit 18 14, (2001).</li><li>Morales, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+and+p53+synergize+to+suppress+genomic+instability+and+lymphomagenesis.">53BP1 and p53 synergize to suppress genomic instability and lymphomagenesis.</a> Proc Natl Acad Sci USA. 103 (9), 3310-3315 (2006).</li><li>Halaby, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+interaction+of+Rnf8+and+p53+in+the+protection+against+genomic+instability+and+tumorigenesis.">Synergistic interaction of Rnf8 and p53 in the protection against genomic instability and tumorigenesis.</a> PLoS Genet. 9 (1), e1003259 (2013).</li></ol>

Los autores no tienen intereses en conflicto.

Mientras que los linfocitos B activados son particularmente adecuados para la preparación de extensiones de cromosomas mitóticos, también se pueden utilizar otros tipos de células. Linfocitos T compartir muchas de las ventajas de las células B, ya que pueden ser purificados a partir de los nodos de bazo o de los ganglios, y tienen un alto índice mitótico cuando son estimuladas por el crecimiento en medio que contiene mitógenos apropiados 8. Las células madre embrionarias (células ES) también son ad...

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.&#160;Table 1 (the list of solutions to prepare) was omitted from the Protocol section. The contents of Table 1 are as follows:
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solution</td>
			<td>Reagents</td>
			<td>Comments</td>
			<td>Storage</td>
		</tr>
		<tr>
			<td rowspan="3">B Cell Wash Buffer</td>
			<td>500 ml 1x Hanks Balanced Salt Solution</td>
			<td rowspan="3">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="3">4 &#176;C</td>
		</tr>
		<tr>
			<td>5 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td rowspan="8">B Cell Medium</td>
			<td>418.5 ml RPMI-1640</td>
			<td rowspan="8">Filter through 0.22 &#956;m stericup filtration unit</td>
			<td rowspan="8">4 &#176;C</td>
		</tr>
		<tr>
			<td>50 ml 50 &#176;C heat inactivated FCS</td>
		</tr>
		<tr>
			<td>5 ml 100x Pen/Strep</td>
		</tr>
		<tr>
			<td>5 ml glutamine</td>
		</tr>
		<tr>
			<td>5 ml nonessential amino acids</td>
		</tr>
		<tr>
			<td>5 ml sodium pyruvate</td>
		</tr>
		<tr>
			<td>1.8 ml 14.2 M 2-mercaptoethanol</td>
		</tr>
		<tr>
			<td>5 ml 1M HEPES</td>
		</tr>
		<tr>
			<td>Fixative</td>
			<td>3:1 v/v methanol/glacial acetic acid</td>
			<td></td>
			<td>Make fresh</td>
		</tr>
		<tr>
			<td rowspan="2">0.075 M KCl</td>
			<td>1.395 g KCl</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>250 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">FISH Master Mix</td>
			<td>40 ml 50% Dextran sulfate</td>
			<td rowspan="3">Vortex the solution and leave in a shaking platform overnight</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml 20x SSC</td>
		</tr>
		<tr>
			<td>40 ml sterile distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="3">70% Formamide/2x SSC</td>
			<td>10 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7 with 1M HCl</td>
			<td rowspan="3">Aliquot and store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>20 ml distilled H2O</td>
		</tr>
		<tr>
			<td>70 ml deionized formamide</td>
		</tr>
		<tr>
			<td rowspan="2">0.01 M HCl</td>
			<td>1 ml 1M HCl</td>
			<td rowspan="2">Adjust to pH 2.0</td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>99 ml distilled H2O</td>
		</tr>
		<tr>
			<td rowspan="2">1x PBS/MgCl2</td>
			<td>50 ml 1M MgCl2</td>
			<td rowspan="2"></td>
			<td rowspan="2">Room Temperature</td>
		</tr>
		<tr>
			<td>950 ml 1x PBS</td>
		</tr>
		<tr>
			<td rowspan="3">50% Formamide/2x SSC</td>
			<td>20 ml 20x SSC</td>
			<td rowspan="3">Adjust to pH 7.25 
			Formamide used in this step does not have to be deionized.</td>
			<td rowspan="3">Make fresh</td>
		</tr>
		<tr>
			<td>80 ml distilled H2O</td>
		</tr>
		<tr>
			<td>100 ml formamide</td>
		</tr>
		<tr>
			<td rowspan="3">4x SSC/0.1% Tween20</td>
			<td>50 ml 20x SSC</td>
			<td rowspan="3"></td>
			<td rowspan="3">Room Temperature</td>
		</tr>
		<tr>
			<td>200 ml distilled H2O</td>
		</tr>
		<tr>
			<td>250 &#956;l Tween20</td>
		</tr>
		<tr>
			<td rowspan="2">DAPI staining solution (80 ng/ml)</td>
			<td>40 &#956;l 0.2mg/ml DAPI stock</td>
			<td rowspan="2"></td>
			<td rowspan="2">4 &#176;C in light protected staining jar</td>
		</tr>
		<tr>
			<td>100 ml 2x SSC</td>
		</tr>
		<tr>
			<td rowspan="5">Mowiol Mounting Medium</td>
			<td>6 g glycerol</td>
			<td rowspan="5">Incubate at 50 &#176;C with stirring for 2hrs</td>
			<td rowspan="5">Aliquot and store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>2.4 g Mowiol 4-88</td>
		</tr>
		<tr>
			<td>6 ml distilled H2O</td>
		</tr>
		<tr>
			<td>12 ml 0.2 M TrisCl pH 8.5</td>
		</tr>
		<tr>
			<td>230 &#956;l 1% Thimerosal (w/v in water)</td>
		</tr>
	</tbody>
</table>
Table 1. List of solutions.&#160;

A correction was made to: Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH.

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

El cáncer es causado por la acumulación de mutaciones que afectan a genes que regulan el crecimiento celular normal. La mutación es una consecuencia de cambios en la estructura y la secuencia del genoma causado por el daño al ADN. Daño del ADN puede ocurrir a través de una variedad de procesos, incluyendo agentes exógenos tales como la radiación ionizante y, como un subproducto del metabolismo celular normal, tales como desaminación espontánea de bases de nucleótidos o daños que se producen por el contacto con especies reactivas de oxígeno 1. Aunque las células de mamíferos poseen una gama de actividades de reparación que puede revertir el daño de ADN o restaurar la secuencia en los sitios de ruptura, sin embargo, las mutaciones se acumulan durante toda la vida de una célula. Daño del ADN puede contribuir además a senescencia y pérdida de potencia de las células madre, dos procesos que están asociados con la enfermedad de envejecimiento asociado 2. La comprensión de la reparación del daño en el ADN es, por tanto, de vital importancia en el tratamiento de dos signocuestiones ificant en la salud pública. La evidencia creciente sugiere que las vías de reparación del ADN de mamíferos pueden contribuir a la evolución del genoma de células de cáncer 3,4, lo que hace aún más imprescindible para entender los procesos implicados en la supresión de la mutación a nivel molecular. La visualización directa de las aberraciones cromosómicas es un potente y cuantitativos medio de determinar el grado de inestabilidad genómica en un tipo de célula particular. Cromosomas condensados ​​a partir de células en metafase se pueden aislar e inspeccionados usando luz o microscopía fluorescente. Tales enfoques citogenéticos han sido en la práctica desde hace varias décadas y se pueden utilizar para demostrar la aparición de translocaciones o tipos específicos de aberraciones cromosómicas asociadas con la pérdida de las actividades de reparación del ADN. El protocolo se presta a varias prórrogas posibles: los cromosomas se pueden marcar con sondas para cariotipo espectral (SKY) o fluorescente multicolor de hibridación in situ(MFISH) para identificar las translocaciones 5,6. Estas técnicas permiten también la frecuencia de las translocaciones cromosómicas y la estructura de las translocaciones cromosómicas complejas por determinar, que proporciona información adicional más allá de lo que es posible con este protocolo. Alternativamente, las sondas específicas de secuencia pueden ser generados y utilizados para probar la frecuencia de rotura del ADN en sitios genómicos seleccionados 7. En este protocolo, se describe la preparación de los cromosomas en metafase se propaga a partir de linfocitos B. Se utiliza un ácido nucleico (PNA) péptido-sonda marcada con fluorescencia para las repeticiones teloméricas, que marca de manera eficiente los telómeros en el cromosoma en metafase se extiende Este protocolo tiene varias ventajas. Las células B pueden ser inducidas a crecer a alto índice mitótico de modo que los diferenciales de alta calidad consistentemente pueden ser producidos. Células B de ratones genéticamente modificados son también mucho menos probable que contenga mutaciones genéticas secundarias que pueden confundir el análisis de la contribuciónde genes específicos a la integridad genómica. El enfoque PNA-FISH se puede completar en un día, y permite anotar más precisa de roturas cromosómicas. Mediante el uso de este enfoque, sobre todo en combinación con el equipo específico que se describe en este protocolo, es posible producir, los diferenciales de alta calidad muy consistentes y analizar rápidamente la velocidad y el tipo de inestabilidad genómica.

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rapid analysis of chromosome aberrations in mouse b lymphocytes by pna-fish

Reparación del ADN defectuoso provoca un aumento de la inestabilidad genómica, que es la causa raíz de las mutaciones que conducen a la tumorigénesis. Análisis de la frecuencia y tipo de aberraciones cromosómicas en diferentes tipos de células permite defectos en las vías de reparación del ADN no se ha dilucidado. Entender la biología de reparación del ADN de los mamíferos se ha ayudado en gran medida por la producción de ratones con nocauts en genes específicos. El objetivo de este protocolo es cuantificar la inestabilidad genómica en los linfocitos B de ratón. Etiquetado de los telómeros con sondas PNA-FISH (ácido nucleico peptídico - hibridación fluorescente in situ) facilita el análisis rápido de la inestabilidad genómica en las extensiones de cromosomas en metafase. Células B tienen ventajas específicas relativas a los fibroblastos, porque tienen la ploidía normal y un índice mitótico superior. Cultivo a corto plazo de las células B, por tanto, permite la medición precisa de la inestabilidad genómica en una población celular primaria que es probable que tenga mutación genética secundaria menoss de lo que se encuentra típicamente en fibroblastos transformados o líneas de células de pacientes.

Los cromosomas en metafase de una célula derivados deben formar un clúster discreta que contiene 40 cromosomas (si usando células de ratón) (Figura 1A). Cada cromosoma tiene un centrómero en un extremo, que es visible como una esfera de color azul claro. En cromosomas normales, dos señales de los telómeros son vistos en el extremo de las cromátidas y dos señales por cada centrómero. Núcleos en interfase estarán presentes en la diapositiva también. Estos serán visibles como esferas azul, co...

Watch this Scientific Journal Video about Rapid Analysis of Chromosome Aberrations in Mouse B Lymphocytes by PNA-FISH at JoVE.com

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

Vías de reparación del ADN son esenciales para el mantenimiento de la integridad genómica y la prevención de la mutación y el cáncer. El objetivo de este protocolo es cuantificar la inestabilidad genómica mediante la observación directa de las aberraciones cromosómicas en metafase se propaga a partir de células B de ratón utilizando hibridación in situ fluorescente (FISH) para las repeticiones teloméricas de ADN.

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 ...

rapid-analysis-chromosome-aberrations-mouse-b-lymphocytes-pna

Research

JoVE Journal

Immunology and Infection

16.8K vistas.  Rutgers, the State University of New Jersey.  Vías de reparación del ADN son esenciales para el mantenimiento de la integridad genómica y la prevención de la mutación y el cáncer. El objetivo de este protocolo es cuantificar la inestabilidad genómica mediante la observación directa de las aberraciones cromosómicas en metafase se propaga a partir de células B de ratón utilizando hibridación in situ fluorescente (FISH) para las repeticiones teloméricas de ADN. 

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

Combinación de adhesivo basado en cinta de muestreo y de fluorescencia In situ La hibridación para la detección rápida de Salmonella En los productos frescos

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Investigación

Inmunología e Infección

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as 
insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during 
the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the 
radionuclide Chromium 51 (51Cr) to label target cells. These targets are then exposed to effector cells and the release of 51Cr from target 
cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of 51Cr.
 As effector cells, we used an activated autoreactive clonal population of CD8+ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with 51Cr labeled target NIT cells for 16 hours, release of 51Cr was recorded to calculate specific lysis 
Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37&deg;C, with 5% CO2, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.
Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Métodos para evaluar la Beta mediada por la muerte celular por linfocitos T citotóxicos

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease.  During the pathogenesis, patients become progressively more insulinopenic as 
insulin ...

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Aislamiento de linfocitos de ratón del tracto genital Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Análisis rápido y robusto de Celular y Molecular Polarización Inducida por Chemokine Señalización

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

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.

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 ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

Un protocolo simple y rápida para no enzimáticamente disociarse Tejidos Humanos frescas para el Análisis de los linfocitos infiltrantes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

Protocolo Aislamiento eficiente para B y linfocitos T a partir Humanos Palatine amígdalas

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

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 ...

Molecular Diffusion in Plasma Membranes of Primary Lymphocytes Measured by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial and temporal resolution. FCS can quantify the molecular concentration and diffusion coefficient of fluorescently labeled molecules in the cell membrane. This technique has the ability to explore the molecular diffusion characteristics of molecules in the plasma membrane of immune cells in steady state (i.e., without processes affecting the result during the actual measurement time). FCS is suitable for studying the diffusion of proteins that are expressed at levels typical for most endogenous proteins. Here, a straightforward and robust method to determine the diffusion rate of cell membrane proteins on primary lymphocytes is demonstrated. An effective way to perform measurements on antibody-stained live cells and commonly occurring observations after acquisition are described. The recent advancements in the development of photo-stable fluorescent dyes can be utilized by conjugating the antibodies of interest to appropriate dyes that do not bleach extensively during the measurements. Additionally, this allows for the detection of slowly diffusing entities, which is a common feature of proteins expressed in cell membranes. The analysis procedure to extract molecular concentration and diffusion parameters from the generated autocorrelation curves is highlighted. In summary, a basic protocol for FCS measurements is provided; it can be followed by immunologists with an understanding of confocal microscopy but with no other previous experience of techniques for measuring dynamic parameters, such as molecular diffusion rates.

A method to measure protein diffusion in membranes of primary immune cells using fluorescence correlation spectroscopy (FCS) is described. In this paper, the use of antibodies for fluorescent labeling is illustrated.

Difusión molecular en el plasma membranas de linfocitos primarios medidos por espectroscopia de correlación de fluorescencia

Fluorescence correlation spectroscopy (FCS) is a powerful technique for studying the diffusion of molecules within biological membranes with high spatial ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

La cuantificación rápida de Blastogénesis inducida por mitógeno de linfocitos T para identificar fármacos inmunomoduladores

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...