Apoyado en parte por una subvención del Instituto Nacional del Cáncer 1R03CA172923.

Declaración de Ética: El uso de ratones en la investigación sólo es aceptable en los casos en que podría avanzar en la comprensión humana de la biología fundamental o hacia el eventual tratamiento de la enfermedad. 1. Orden <ol><li> Compra hembra C57BL / 6 ratones (edad de 6 - 8 semanas), 5 ratones por grupo. Se requieren varios días para los ratones para ajustar. </li></ol> 2. Preparación de las células B16-BL6 <ol><li> Retire las placas de cultivo celular de incubadora a 37 grados y coloque en una campana estéril. Las placas deben ser aproximadamente 70% confluentes con células de melanoma murino B16-BL6. A mayor confluencia puede alterar las células potencial metastásico. </li><li> Añadir 1 ml de 0,05% tripsina-EDTA por placa, dejar reposar durante 1 minuto, y luego aspirar la solución de tripsina-media. </li><li> Añadir 1 ml de tripsina por placa y las placas de volver a la incubadora durante 10 min. </li><li> Después de mover las placas de la incubadora a la campana estéril, añadir 4 ml de suero libre de Rosewell Parque Memorial Institute Medium (RPMI) a cada placa. </li><li> Recoger las células con una pipeta estéril, motorizado. Expulsar células contra la parte inferior de la placa, con la punta presionado en un muy ligero ángulo, para romper las células. Repetir varias veces con el fin de asegurar la separación adecuada de los grupos de células, que de otro modo podrían obstruir la aguja de inyección. Recordar y transferir a un tubo cónico de 50 ml. </li><li> Use un hemocitómetro para determinar la densidad celular. Mantener el número total de células B16-BL6 constante, determinar el volumen de RPMI libre de suero necesario para alcanzar concentraciones finales de 0, 0,065, 0,015, 0,25, y 0,5 millones de células / ml. </li><li> Igualmente alícuota los medios de comunicación en el tubo de 50 ml a 15 ml tubos cónicos. Centrifugar los tubos cónicos a 2.250 xg durante 5 min. Decantar los medios de comunicación. Añadir un volumen apropiado de RPMI libre de suero y confirmar la densidad celular deseada a través de hemocitómetro. Ajuste densities- añadiendo RPMI adicional o hilado y resuspensión en un volumen-más pequeño en consecuencia. </li><li> Alícuotas 500 lde cada suspensión celular en 1,8 ml etiquetados tubos en hielo. </li></ol> 3. vena de la cola de inyección <ol><li> Tome un ratón, que tuvo lugar por la cola, y lo guiará trasera por primera vez en el limitador del ratón. Con el torso en la cámara principal del retenedor, con la cola fuera de ella, tira del ratón hacia el final del inmovilizador. Inserte el limitador-émbolo, sin dejar de empujar hasta que el ratón es seguro. </li><li> Girar el ratón 90 grados de tal manera que una de las venas de la cola lateral esté orientado hacia arriba. El uso de una almohadilla con alcohol, limpie vigorosamente el lado de la cola del ratón, en particular cuando la vena de la cola es más visible. Nota: Una buena iluminación es esencial para la visualización de la vena de la cola efectiva en 6J / B57BL. Las lámparas de calor o almohadillas de cirugía climatizada, aunque no es necesario, también ayudan a aumentar el volumen de la vena de la cola. </li><li> En una campana de cultivo estéril, recoger 300 l de medio de cultivo celular del tubo de 2 millones de células / ml. Invertir la aguja, moviendo los side y empujando el émbolo, para expulsar las burbujas de la jeringa. Expulsar cultivo RPMI para dar lugar a un volumen final de 250 l. </li><li> Extender el ratón de cola con la mano no dominante. Mantenga la cola con el extremo proximal de la cola elevada con el dedo índice de la mano no dominante, y la parte distal de la cola deprimido con el pulgar. Nota: En este modo, la vena de la cola se mantiene en una posición lateral, paralela a la superficie de contención, con una entrada congruente posible en la parte más distal de la cola. </li><li> Insertar la aguja en la vena de la cola, hacia el extremo distal, en un ángulo descendente minutos para empezar, y luego ajustar la aguja para que coincida con el ángulo de la vena de la cola a la inserción adicional (bajando extremo del émbolo de la jeringa respecto a la aguja) . </li><li> Insertar la aguja aproximadamente 1 cm en la vena de la cola. Después de la inserción, comienzan a empujar el émbolo, se mantiene la aguja constante. Nota: Si la aguja está en la vena de la cola y el extremo de la aguja es not obstruido los medios de comunicación debe fluir sin obstáculos en la vena. inyección eficaz se caracteriza por depuración de la sangre de la vena. Si hay resistencia, o una burbuja empieza a formar en el lugar de la inyección, retire la aguja e inténtelo de nuevo en una ubicación más proximal a lo largo de la vena de la cola. </li><li> Retire la aguja de la vena y lo descarta. Mantenga una gasa contra el sitio de entrada para detener cualquier sangrado. </li><li> Retire el émbolo del retenedor y colocar el puntero del ratón en la jaula destinatario. </li><li> Repita este procedimiento para todas las demás concentraciones. Nota:. Pulmón establecimiento focos tarda aproximadamente 2 - 3 semanas, con una variabilidad dependiendo de la línea celular y el tamaño deseado de los focos 9 En el ínterin, los ratones deben ser monitorizados para detectar síntomas que podrían justificar la eutanasia temprano, como jadeo excesivo. </li></ol> 4. Contar el pulmón Focos <ol><li> La eutanasia el ratón mediante la colocación de una cámara de CO 2 y la exposición al 100% de CO2 </sub&gt; introdujo en 10 a 30% de volumen de la cámara por minuto durante 5 minutos. Siga con una dislocación cervical. </li><li> Splay el ratón sobre la espuma de poliestireno. Usando tijeras quirúrgicas, crear una incisión quirúrgica a lo largo del esternón. </li><li> Haga dos incisiones adicionales, tanto de la parte superior de la incisión del esternón y la ramificación hacia los hombros del ratón, la exposición de la caja torácica. </li><li> Hacer dos cortes, cada uno a lo largo de los lados laterales del esternón, para eliminar de la caja torácica del torso, la exposición de los pulmones y el corazón. </li><li> Sosteniendo el corazón con pinzas quirúrgicas, cortar la tráquea y el esófago, liberando el corazón y los pulmones del ratón. Colocar debajo de un microscopio de disección, un aumento de 10x, para una mejor visualización. </li><li> Diseccionar el corazón de los pulmones y retire el timo, dejando sólo los dos pulmones. </li><li> Con los pulmones bajo un microscopio de disección, comenzar a contar los focos de pulmón. Cada focos debe aparecer como una intromisión circular, de color marrón en la superficie de los pulmones. <ol><li> porla coherencia entre los ratones, contar el número de focos en la superficie de un lóbulo, y luego invertir ese lóbulo. Hacer cada uno de los cuatro lóbulos permite individualmente para el recuento más fácil. </li></ol></li><li> Guardar y fijar los pulmones si se realiza IHC, de lo contrario, desechar los restos. </li></ol>

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F., 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=Improved+survival+with+Ipilimumab+in+patients+with+metastatic+melanoma.">Improved survival with Ipilimumab in patients with metastatic melanoma.</a> NEJM. 363 (13), 711-723 (2002).</li><li>Ekwueme, D. U., Guy, G. P., Li, C., Rim, S. H., Parelkar, P., Chen, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+health+burden+and+economic+costs+of+cutaneous+melanoma+motality+by+race/ethnicity-United+States.">The health burden and economic costs of cutaneous melanoma motality by race/ethnicity-United States.</a> J. Am. Acad. Dermatol. 65, 133-143 (2011).</li><li>Alizadeh, A. 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Los autores declaran que no tienen intereses financieros en competencia.

Las células tumorales circulantes de una inyección en vena de cola representar las metástasis que evaden sitios de crecimiento primario y migrar a través de rutas como el torrente sanguíneo o el sistema linfático, de vez en cuando establecerse en los lechos capilares de lugares distantes. 14 el protocolo descrito anteriormente sirve como un modelo para secundaria metástasis. Con un número óptimo de células B16-BL6 inyectado, los experimentadores pueden determinar el efecto relativo de un fármaco administrado o población de células inmunes, post-neutralización, sobre la metástasis de células de cáncer. Este protocolo tiene sus limitaciones. Las células tumorales circulantes se han seleccionado por su capacidad de metástasis y por lo tanto son no inmunogénicos, que tiene menores niveles de mayor histocompatibilidad de clase 1 moléculas. 12 Además, la introducción repentina y agrupada de gran número de metástasis es a diferencia de la situación típica en pacientes donde un pequeño número de células tumorales metastásicasdisipar durante un período prolongado de tiempo de la localización del tumor primario. Además, las células tumorales por vía intravenosa introducidas son de cultivo de tejidos y no de un origen del tumor primario. Por lo tanto, estas células son a diferencia de las metástasis secundarias que resultan de alteraciones en la adhesión celular, la migración, el reconocimiento inmune y el receptor establecimiento de órganos. 15,16 Un protocolo alternativo sería la inyección subcutánea de B16 / BL6 para la generación de tumores primarios y el análisis de los resultantes metástasis pulmonares &quot;espontáneas&quot;. Se han encontrado estas metástasis &quot;espontáneas&quot; para contener una mayor heterogeneidad que sus contrapartes &quot;experimentales&quot;, más muy similares a los de los pacientes humanos. El protocolo es limitante en su capacidad para determinar la influencia de un compuesto experimental sobre la metástasis. Con una inyección vena de la cola lateral, el número de células de melanoma inyectadas en el torrente sanguíneo se puede aproximar a través de hemocytometer. Con la metástasis &quot;espontánea&quot;, sin embargo, hay una mayor heterogeneidad entre los tumores, la adición de una variable adicional para el número de focos de pulmón resultante. 16 En conclusión, el modelo de melanoma murino B16-BL6 vena de la cola representa un modelo sencillo para determinar la influencia de un tratamiento o la inmunoterapia en la metástasis. Por vía intravenosa la inyección de células tumorales circulantes, los investigadores pueden replicar, en cierta medida, los pacientes post-metástasis. Teniendo en cuenta los efectos destructivos de células tumorales metastásicas en los pacientes de cáncer, este modelo es una herramienta poderosa para la comprensión del proceso de múltiples etapas de la metástasis.

La metástasis es una causa importante de muerte entre los pacientes con tumores malignos. El proceso de la diseminación metastásica de las células cancerosas es poco conocida pero parece implicar varios pasos, incluyendo la invasión de los tejidos adyacentes, intravasación en los vasos linfáticos y la vasculatura, la supervivencia y la translocación dentro del sistema circulatorio, la extravasación de la vasculatura en el lugar de la metástasis, la adaptación al nuevo microambiente del nuevo sitio, y la colonización en el nuevo sitio de la proliferación y la formación de tumores secundarios. 1 Cada uno de estos eventos biológicos implican la transformación, la difusión y la supervivencia de las células tumorales metastásicas. Por ejemplo, la transformación del fenotipo epitelial de la célula tumoral en un fenotipo mesenquimal, junto con la interacción exitosa con la matriz extracelular, que puedan invadir los tejidos adyacentes y metastatizar a otras partes del cuerpo. 2,3 Además, estos metastásico Célulasdebe sobrevivir en el torrente sanguíneo circulante y evadir la vigilancia inmune del huésped. 4,5 Por último, cuando se implanta en un sitio distante dentro del cuerpo, las células tumorales deben adaptarse a su microambiente con el fin de proliferar y formar tumores secundarios. 6,7 Por lo tanto , a pesar de la metástasis es un fenómeno común entre los pacientes con cáncer, las células tumorales metastásicas tienen múltiples áreas de vulnerabilidad que son susceptibles de intervención terapéutica. Dada la naturaleza inmunogénica de melanoma, y el reciente interés en inmunoterapias, modelos para el melanoma son cada vez más útil. 8 Sólo en los Estados Unidos, el melanoma es la causa de un estimado de 9.000 muertes al año. 9 lugares comunes de metástasis de melanoma son los huesos, el cerebro , el hígado y los pulmones. La mayoría de las metástasis a sitios distantes se producen a través del torrente sanguíneo. Las células tumorales circulantes en la sangre puede eludir aclaramiento inmunitario, llegar a un lecho capilar de un órgano distal yinvadir a través de las células endoteliales de los vasos sanguíneos con el fin de establecer con éxito a sí mismos. 4-7,10, 11 Para imitar los fenómenos comunes y virulentas de metástasis, se ha creado la línea de células B16-BL6 murino. A difunto de la C57BL / 6 línea celular de melanoma parental B16-F0, B16-BL6 es el producto final de 10 selecciones sucesivas de metástasis de pulmón de la inyección intravenosa (que resulta en B16-F10), seguido de 6 selecciones sucesivas de penetración de la membrana de la vejiga. 12 Como tal, es una línea de células de melanoma fiable para el establecimiento de focos metastásicos en el ratón, sobre todo cuando se inyecta por vía intravenosa. 13 Después de la inyección de una cantidad suficiente de B16-BL6 células en la vena de la cola de un ratón 6 / B57BL, seguido de dos o más semanas para permitir la implantación y el crecimiento de las células B16 / BL6, focos metastásicos se formará en los pulmones. Tras la eutanasia y la inspección por microscopía de disección, el número defocos individuales presentes se puede cuantificar. Esto, a su vez, puede ser usado para establecer un efecto dosis-metástasis, como el número de células B16-BL6 inyectados se correlaciona con el número de focos formados en la superficie de los pulmones. Este modelo es conocido como &quot;la metástasis experimental&quot;, donde se introducen directamente las células conocidas metastásico en el torrente sanguíneo, lo que facilita una rápida dispersión y establecimiento y predecible en los pulmones, el hígado, u otro órgano de investigación. Esto está en contraste con la &quot;metástasis espontánea&quot;, donde se implantan células tumorales, a menudo por vía subcutánea, y las metástasis se originan a partir orgánicamente eliminar las células cancerosas. 14,15 Es importante para inyectar una cantidad apropiada de células B16 / BL6 en las venas de la cola de los ratones. Demasiados, y los pulmones serán cubiertos con metástasis y la vecina focos serán indistinguibles unos de otros. Demasiado pocos, y la influencia de un agente terapéutico será indiscernible debido a vari inherenteciones entre las inyecciones. Con el fin de obtener un número óptimo de focos en los pulmones de los ratones C57BL / 6, es necesario correlacionar el número de células inyectadas con la cantidad de focos metastásicos establecido sobre la superficie de los pulmones, teniendo en cuenta la distinguibilidad de individuo focos. Este protocolo demostrará un modelo de melanoma murino metastásico de ratones C57BL / 6.

<table border="0" cellpadding="0" cellspacing="0" width="1227">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:377px;">Trypsin</td>
			<td style="width:275px;">Corning</td>
			<td style="width:288px;">MT25052CV</td>
			<td style="width:287px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium</td>
			<td>Corning</td>
			<td>MT15040CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phase Contraast Hemacytometer</td>
			<td>Hausser</td>
			<td>02-671-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro-Fine IV Insulin Syringes</td>
			<td>BD</td>
			<td>14-829-1D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pads</td>
			<td>Fisherbrand</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse Restrainer</td>
			<td>Braintree Scientific</td>
			<td>NC9999969</td>
			<td>Restrainer choice depends on age/size of mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heating Pad</td>
			<td>Harry Schein</td>
			<td>NC0012697</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

injection of syngeneic murine melanoma cells to determine their metastatic potential in the lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Tras una inspección visual, la variación en los focos de la superficie es evidente. Contar el número de tumores en virtud de un microscopio de disección debe producir una relación lineal entre el número de células tumorales inyectadas y el número de focos resultante y contable. El uso de un número óptimo de células inyectables, el objetivo es una donde los pulmones no están completamente cubiertas, ya que están en 500.000 células / ml en la Figura 1A, y que no están cubiertos demasiado escasa, ya que están en 62.500 células / ml y 125.000 células / ml. Esto también es cuantificable por correlación lineal (Figura 1B). <img alt="Figura 1" src="/files/ftp_upload/54039/54039fig1.jpg" /> Figura 1. Los focos de pulmón resultante de vena de la cola inyectaron células B16 / BL6. (A) focos de pulmón puede ser visualizado en la superficie de los pulmones de ratones C57BL / 6 como masas circulares marrones (focos representante en virtud de las flechas). Como los antrosdad del cultivo celular inyectada (por encima de cada imagen) aumenta, también lo hace el número correspondiente de resultante focos de pulmón (por debajo de cada imagen). Los pulmones del ratón con la mayor densidad de células inyectadas tiene la mayor cobertura visual de focos de pulmón. Las barras de escala representan 20 mm. (B) Enumeración de los focos de pulmón en relación con la densidad de cultivo de células. Existe una relación aproximadamente lineal por encima de 125.000 células / ml, según lo determinado por hemocitómetro. <a href="https://www.jove.com/files/ftp_upload/54039/54039fig1large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a>

Watch this Scientific Journal Video about Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs at JoVE.com

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

La inyección de células de melanoma murino singénico para determinar su potencial metastásico en los pulmones

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

injection-syngeneic-murine-melanoma-cells-to-determine-their

Research

JoVE Journal

Medicine

17.5K vistas.  Beth Israel Deaconess Medical Center & Harvard Medical School. Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

Video: Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Un In Vitro Sistema para el Estudio de latencia del tumor y el salto hacia el crecimiento metastásico

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Investigación

Medicina

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

La medición y tratamiento de la represión en la ambliopía

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Usando cuantitativa en tiempo real PCR para determinar quimerismo de células donantes en un modelo competitivo Trasplante de médula ósea murina

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

Infarto de miocardio y la evaluación de resultados funcionales en los cerdos

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

Un Modificado<em&gt; In vitro</em&gt; Invasión Ensayo para determinar el papel potencial de hormonas, citoquinas y / o factores de crecimiento en la invasión de células del cáncer de Mediación

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Enfoque Formación: Un análisis basado en células para determinar el potencial oncogénico de un gen

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Measurement of the Pressure-volume Curve in Mouse Lungs

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential phenotypic changes are best assessed by measurement of the changes in lung elasticity. To best understand specific mechanisms underlying such pathologies in mice, it is essential to make functional measurements that can reflect the developing pathology. Although there are many ways to measure elasticity, the classical method is that of the total lung pressure-volume (PV) curve done over the whole range of lung volumes. This measurement has been made on adult lungs from nearly all mammalian species dating back almost 100 years, and such PV curves also played a major role in the discovery and understanding of the function of pulmonary surfactant in fetal lung development. Unfortunately, such total PV curves have not been widely reported in the mouse, despite the fact that they can provide useful information on the macroscopic effects of structural changes in the lung. Although partial PV curves measuring just the changes in lung volume are sometimes reported, without a measure of absolute volume, the nonlinear nature of the total PV curve makes these partial ones very difficult to interpret. In the present study, we describe a standardized way to measure the total PV curve. We have then tested the ability of these curves to detect changes in mouse lung structure in two common lung pathologies, emphysema and fibrosis. Results showed significant changes in several variables consistent with expected structural changes with these pathologies. This measurement of the lung PV curve in mice thus provides a straightforward means to monitor the progression of the pathophysiologic changes over time and the potential effect of therapeutic procedures.

Here we present a protocol to simply and reliably measure the lung pressure-volume curve in mice, showing that it is sufficiently sensitive to detect phenotypic parenchymal changes in two common lung pathologies, pulmonary fibrosis and emphysema. This metric provides a means to quantify the lung&#8217;s structural changes with developing pathology.

Medición de la curva de presión-volumen en mouse Pulmones

In recent decades the mouse has become the primary animal model of a variety of lung diseases. In models of emphysema or fibrosis, the essential ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Un &quot;paciente-Like&quot; ortotópico Singénico modelo de ratón de carcinoma hepatocelular Metástasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Aislamiento de células progenitoras endoteliales de voluntarios sanos y su potencial migratorio influenciado por las muestras de suero después de cirugía cardiaca

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...