Esta investigación fue financiada en parte por el Programa de Investigación Intramural del Instituto Nacional del Cáncer de los Institutos Nacionales de Salud, y por el NCI concede UA5CA152907.

1. Colección de medio condicionado a la Prueba de Potencial angiogénico <ol><li> Se cultivan las células primarias o inmortalizadas a ensayar para poder angiogénico y anti-angiogénico en medio de suero nativas o bajas y recoger el medio condicionado. <ol><li> Alternativamente, el uso de medios acondicionados de inmediato, o se dividió en alícuotas y se almacenó a -80 ° C durante varios meses. Utilizar los medios de suero nativa o bajo nonconditioned como un control negativo, y el uso de medios no acondicionado completa de crecimiento (10% de FBS, o la concentración apropiada) como control positivo. Alternativamente, para probar posibles estimuladores de la angiogénesis, suplemento de carbón despojado medios que carecen de factores de crecimiento con concentraciones conocidas de proteínas específicas de interés y se compara con el factor de crecimiento medios de comunicación libres por sí solos. </li></ol></li></ol> 2 Preparación de las células endoteliales antes del ensayo <ol><li> Dos días antes del ensayo, el paso 3B-11 células en un nuevo matraz T-75. Número de paso de las células es importante. Este ensayo funciona mejor cuando las células están entre el segundo y sexto pasajes. El ensayo no va a funcionar bien si las células endoteliales se utilizan antes de la segunda o después del décimo paso. </li><li> Se cultivan las células durante 24 horas en DMEM suplementado con 10% de FBS, 2 mM L-glutamina, piruvato de sodio 1 mM, 100 U / ml de penicilina, y 100 mg / ml de estreptomicina. </li><li> Un día antes del ensayo, el suero de hambre células 3B-11 como sigue: <ol><li> Aspirar los medios de comunicación de las células 3B-11. </li><li> Añadir un medio de suero reducido de DMEM suplementado con 0,2% de FBS, 2 mM L-glutamina, piruvato de sodio 1 mM, 100 U / ml de penicilina, y 100 mg / ml de estreptomicina. </li><li> Se cultivan las células durante 24 h más. </li></ol></li></ol> 3. Preparación de los reactivos antes del ensayo NOTA: Las concentraciones de BME son muy variables dependientes en el lote. BME no funciona bien si la concentración es menor que 10 mg / ml, por lo tanto, el fabricante BME debe ser contactado antes de la compra para garantizaradquisición de un lote de concentrado adecuadamente. <ol><li> Descongelar reduce el factor de crecimiento BME en el refrigerador. BME se solidifica cuando se calienta, por lo que debe mantenerse en frío antes de su uso. BME se puede almacenar en el refrigerador por un par de semanas, o en alícuotas y se congeló a -80 ° C. </li><li> Un factor de crecimiento reducido BME se prefiere sobre el tradicional BME. La falta de factores angiogénicos en el BME, será más fácil observar el efecto que los factores a partir de los medios condicionados tienen en la formación del tubo. </li><li> Descongele los medios de comunicación a ensayar. </li></ol> 4 Preparación de las células endoteliales Inmediatamente antes del ensayo <ol><li> Lavado 3B-11 células en DPBS. <ol><li> Si se desea la visualización con fluorescencia o microscopía confocal, antes de lavar las células 3B-11, añadir calceína AM a las células endoteliales en los medios a una concentración final de 2 mg / ml. Coloque células marcadas calceína en la oscuridad a 37 ° C y 5% de CO 2 antes del inicio del ensayo. Disolver de stock calceınaAM en DMSO y una vez calceína se diluye en concentración final de DMSO medios de comunicación no debe exceder de 0,1%. Después de 30 a 45 min de lavado calceína AM de las células (paso 4.1) y continuar con el experimento siguiente. </li></ol></li><li> Tripsinizar las células mediante la adición de 1 ml de tripsina-EDTA al matraz T-75. </li><li> Después de tripsinización es completa, neutralizar la tripsina resuspendiendo las células a un volumen final de 10 ml en DMEM, 10% FBS, 2 mM L-glutamina, piruvato de sodio 1 mM, 100 U / ml de penicilina, y 100 mg / ml de estreptomicina. </li><li> Las células de filtro a través de filtro de células 100 micras para eliminar grumos. </li><li> Contar las células y volver a suspender las células a una concentración final de 7,5 x 10 6 células / ml en DMEM, 10% FBS, 2 mM L-glutamina, piruvato de sodio 1 mM, 100 U / ml de penicilina, y 100 mg / ml de estreptomicina. </li></ol> Ensayo de Formación 5. Tubo <ol><li> Coloque la placa de 24 pocillos y 1 ml de recomendaciones en el refrigerador o en hielo durante 20-30 minutos antes de cargar la placa para que el BME no Prematurely solidificar. </li><li> Pipetear 250 l de factor de crecimiento reducido BME en cada pocillo de una placa de 24 pocillos. Evitar la creación de burbujas por no presionar la pipeta hasta la última parada, mientras que la dispensación. Tenga cuidado de que suficiente BME se mantiene en el centro del pozo cuando se forma el menisco; si la matriz es demasiado delgada, las células sólo se forman una monocapa. </li><li> Incubar la placa de 24 pocillos a 37 ° C y 5% de CO 2 durante 30 minutos para solidificar BME. </li><li> Una vez que BME ha puesto, mezcle aproximadamente 300 l de medio condicionado con 10 l de resuspendidas 3B-11 células (aproximadamente 75.000 células). <ol><li> Ajustar el volumen de medio acondicionado y número de células sembradas en función del tipo de célula endotelial utilizado, y probar las diluciones seriadas de los medios acondicionados para demostrar la actividad. Si las células que comparan cultivadas bajo diferentes condiciones, normalizar acondicionado volumen de medios de comunicación para el número de células que fueron utilizadas para acondicionar los medios de comunicación. </li></ol></li><li> Tubos will comienzan a formarse dentro de 2-4 horas. Dependiendo de la concentración de factor angiogénico en los medios acondicionados, la formación de pico tubo puede ocurrir entre 3 y 12 horas y el momento del ensayo tendrá que ser optimizado. Si se trabaja con células endoteliales primarias en lugar de células inmortalizadas, la formación inicial del tubo se puede retrasar por varias horas. <ol><li> Tubos a menudo comienzan a deteriorarse dentro de 18 hr y células endoteliales se someterá a la apoptosis. </li></ol></li><li> En el momento de la formación del tubo de pico, medios de comunicación cuidadosamente aspirado desde el pozo para evitar la interrupción de la red en el tubo de BME. <ol><li> Lavar cada pocillo con 1 ml de DPBS, y aspirar. <ol><li> Para la visualización inmediata, añadir 1 ml de DPBS frescas a cada tubo de la red bien y la imagen usando un microscopio invertido conectado a una cámara digital, o fluorescencia / microscopio confocal si las células se tiñeron con calceína AM como en 4.1.1. </li><li> Para arreglar la red de metro para la visualización posterior, y añadir 4% de paraformaldehído a cadaaspirado pocillo e incubar durante 15 min a temperatura ambiente. <ol><li> Eliminar paraformaldehído, lavar dos veces con DPBS, a continuación, añadir 1 ml de DPBS frescos a cada pocillo. </li><li> Microscópicamente imagen antes de la fijación como unos tubos pueden romper durante este procedimiento. </li></ol></li></ol></li></ol></li></ol> 6. Cuantificación de tubo Red <ol><li> Cuantificar la red de metro de diferentes maneras dependiendo de las preferencias del investigador, y varios programas de ordenador tienen capacidad para medir los siguientes parámetros: número de tubos; número de bucles / mallas; número de sitios de sucursal / nodos; longitud de los tubos. </li><li> Utilice programa informático representativo como imagen J con el plugin Angiogénesis analizador 16 para la cuantificación de las redes de metro. </li></ol>

<ol>
	<li>Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+life,+disease+and+medicine.">Angiogenesis in life, disease and medicine.</a> Nature. 438, 932-936 (1038).</li><li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473, 298-307 (2011).</li><li>Potente, M., Gerhardt, H., Carmeliet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+and+therapeutic+aspects+of+angiogenesis.">Basic and therapeutic aspects of angiogenesis.</a> Cell. 146, 873-887 (2011).</li><li>Coultas, L., Chawengsaksophak, K., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+and+VEGF+in+vascular+development.">Endothelial cells and VEGF in vascular development.</a> Nature. 438, 937-945 (2005).</li><li>Bouis, D., Kusumanto, Y., Meijer, C., Mulder, N. H., Hospers, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+on+pro-+and+anti-angiogenic+factors+as+targets+of+clinical+intervention.">A review on pro- and anti-angiogenic factors as targets of clinical intervention.</a> Pharmacological research : the official journal of the Italian Pharmacological Society. 53, 89-103 (2006).</li><li>Ausprunk, D. H., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+and+proliferation+of+endothelial+cells+in+preformed+and+newly+formed+blood+vessels+during+tumor+angiogenesis.">Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis.</a> Microvascular research. 14, 53-65 (1977).</li><li>Chung, A. S., Lee, J., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tumour+vasculature:+insights+from+physiological+angiogenesis.">Targeting the tumour vasculature: insights from physiological angiogenesis.</a> Nature reviews. Cancer. 10, 505-514 (2010).</li><li>Chung, A. S., Ferrara, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+and+pathological+angiogenesis.">Developmental and pathological angiogenesis.</a> Annual review of cell and developmental biology. 27, 563-584 (2011).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis.">Tumor angiogenesis.</a> The New England journal of medicine. 358, 2039-2049 (2008).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis:+therapeutic+implications.">Tumor angiogenesis: therapeutic implications.</a> The New England journal of medicine. 285, 1182-1186 (1056).</li><li>Kubota, Y., Kleinman, H. K., Martin, G. R., Lawley, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laminin+and+basement+membrane+in+the+morphological+differentiation+of+human+endothelial+cells+into+capillary-like+structures.">Role of laminin and basement membrane in the morphological differentiation of human endothelial cells into capillary-like structures.</a> The Journal of cell biology. 107, 1589-1598 (1988).</li><li>Arnaoutova, I., George, J., Kleinman, H. K., Benton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endothelial+cell+tube+formation+assay+on+basement+membrane+turns+20:+state+of+the+science+and+the+art.">The endothelial cell tube formation assay on basement membrane turns 20: state of the science and the art.</a> Angiogenesis. 12, 267-274 (2009).</li><li>Arnaoutova, I., Kleinman, H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+angiogenesis:+endothelial+cell+tube+formation+on+gelled+basement+membrane+extract.">In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.</a> Nature protocols. 5, 628-635 (2010).</li><li>Walter-Yohrling, 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=Murine+endothelial+cell+lines+as+models+of+tumor+endothelial+cells.">Murine endothelial cell lines as models of tumor endothelial cells.</a> Clinical cancer research : an official journal of the American Association for Cancer Research. 10, 2179-2189 (2004).</li><li>Connell, K. A., Edidin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+lymphoid+endothelial+cell+line+immortalized+by+simian+virus+40+binds+lymphocytes+and+retains+functional+characteristics+of+normal+endothelial+cells.">A mouse lymphoid endothelial cell line immortalized by simian virus 40 binds lymphocytes and retains functional characteristics of normal endothelial cells.</a> Journal of immunology. , 144-521 (1950).</li><li>Carpentier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+contribution:+Angiogenesis+Analyzer.">ImageJ contribution: Angiogenesis Analyzer.</a> ImageJ News. , (2012).</li></ol>

Los autores no tienen nada que revelar.

La angiogénesis está implicada en los procesos fisiológicos y patológicos. El estudio de los mecanismos implicados en la angiogénesis requiere el uso de ensayos que recapitular los pasos principales en la angiogénesis. El ensayo de formación de tubo celular endotelial ofrece varias ventajas sobre otros ensayos. Es fácil de configurar, es relativamente barato, produce túbulos en cuestión de horas, y es cuantificable. Además, se puede completar en 24 ó 96 placas de pocillos, y por lo tanto se puede utilizar pa...

La angiogénesis, el desarrollo de nuevos vasos sanguíneos a partir de vasos preexistentes, es vital para una variedad de procesos que incluyen el crecimiento de órganos, el desarrollo embrionario, la cicatrización de heridas y 1-3. Nuevo desarrollo de los vasos sanguíneos, se alinearon por las células endoteliales, el suministro de oxígeno y nutrientes a los tejidos, la promoción de la vigilancia inmunológica de las células hematopoyéticas y eliminar los productos de desecho 2,4. La angiogénesis es de vital importancia durante el desarrollo embrionario y fetal. Sin embargo, este proceso permanece latente en el adulto, excepto en tiempos de cicatrización de heridas, el crecimiento del esqueleto, el embarazo o durante el ciclo menstrual 1-3. En las últimas dos décadas, los mecanismos moleculares que regulan la angiogénesis clave han comenzado a surgir. La angiogénesis es un evento estrictamente regulados, equilibrada por señales pro y antiangiogénicos, que incluyen las integrinas, quimiocinas, angiopoyetinas, agentes de detección de oxígeno, moléculas de unión y los inhibidores endógenos 5. Una vez proangiogenic señales tales como el factor de crecimiento de fibroblastos básico (bFGF), factor de crecimiento endotelial vascular (VEGF), factor de crecimiento derivado de plaquetas (PDGF) y factor de crecimiento epidérmico (EGF) activan los receptores de las células endoteliales de las células endoteliales liberan proteasas para degradar la membrana basal. Las células endoteliales proliferan y migran a continuación, la formación de brotes a una velocidad de varios milímetros por día 6,7. La angiogénesis está asociada con diversas condiciones patológicas, incluyendo cáncer, psoriasis, retinopatía diabética, artritis, asma, trastornos autoinmunes, enfermedades infecciosas, y la aterosclerosis 8-10. Debido a la importancia de la angiogénesis en diversas enfermedades, la comprensión de los genes y las vías que regulan este proceso es fundamental para el diseño de mejores terapias. El ensayo de formación de tubo es un método rápido y cuantitativo para la determinación de genes o rutas implicadas en la angiogénesis. Descrita por primera vez en 1988,los principios subyacentes de este ensayo son que las células endoteliales conservan la capacidad de dividirse y migrar rápidamente en respuesta a las señales angiogénicas 11-13. Además, las células endoteliales son inducidas a diferenciarse y formar estructuras similares a tubos cuando se cultivan sobre una matriz de extracto de membrana basal (BME). Estos tubos contienen un lumen rodeado por las células endoteliales unidas entre sí a través de complejos de unión. La formación del tubo se produce rápidamente con la mayoría de los tubos que forman en este ensayo a las 2-6 horas dependiendo de la cantidad y tipo de estímulos angiogénicos. Varios tipos de células endoteliales pueden ser utilizados para este ensayo incluyendo tanto células primarias y líneas celulares inmortalizadas 14,15. La línea celular utilizada para este artículo fue ratón 3B-11, pero la misma metodología se puede aplicar con otras líneas de células endoteliales tales como SVEC4-10 (ratón) o células endoteliales primarias tales como las células HUVEC (humanos). Dependiendo de la línea celular se utiliza y si el ce endotelialLLS se transforman o no transformadas, tendrá que ser llevado a cabo para identificar el momento ideal necesaria para la formación correcta del tubo de optimización.

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			<td height="76" rowspan="2" style="height:76px;width:339px;">Gibco 0.05% Trypsin-EDTA</td>
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			<td height="95" rowspan="2" style="height:95px;width:339px;">Gibco L-Glutamine 200mM (100X)</td>
			<td rowspan="2" style="width:189px;">Life Technologies</td>
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			<td height="57" rowspan="2" style="height:57px;width:339px;">Gibco Pen Strep</td>
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			<td style="width:189px;">ATCC</td>
			<td style="width:124px;">4-X</td>
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endothelial cell tube formation assay for the in vitro study of angiogenesis

La angiogénesis es un proceso vital para el desarrollo del tejido normal y la curación de heridas, pero también se asocia con una variedad de condiciones patológicas. Usando este protocolo, la angiogénesis se puede medir in vitro de una manera rápida, cuantificable. Células endoteliales primarias o inmortalizadas se mezclan con medio condicionado y se sembraron en la matriz de la membrana basal. Las células endoteliales forman estructuras similares a capilares en respuesta a señales angiogénicas que se encuentran en los medios condicionados. La formación del tubo se produce rápidamente con células endoteliales comenzando a alinearse dentro de 1 hora y túbulos que contienen lumen-comenzando a aparecer dentro de 2 horas. Los tubos pueden ser visualizados utilizando un microscopio invertido de contraste de fase, o las células pueden ser tratadas con calceína AM antes del ensayo y tubos visualizado a través de fluorescencia o microscopía confocal. El número de sitios de sucursal / nodos, bucles / mallas, o el número o la longitud de los tubos formados pueden ser fácilmente cuantificado como una medida de en vitro angiogénesis. En resumen, este ensayo se puede usar para identificar los genes y las vías que están involucrados en la promoción o inhibición de la angiogénesis en una manera rápida, reproducible y cuantitativo.

Ratón 3B-11 células endoteliales fueron sembradas en el factor de crecimiento reducido solidificado BME - en este ensayo, el producto Matrigel se utilizó - y siguió a lo largo del tiempo. Como se muestra en la Figura 1, los factores angiogénicos secretados por cualquiera de los queratinocitos o fibroblastos de ratón son capaces de inducir la formación de tubos con el tiempo. Las células endoteliales migran y se empiezan a formar las ramas pequeñas dentro de 1-2 horas de la siembra. Formación d...

Watch this Scientific Journal Video about Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis at JoVE.com

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

El ensayo de formación de tubo es un método rápido, cuantificable para medir la angiogénesis in vitro. Las células endoteliales se combinan con los medios condicionados y se sembraron en extracto de membrana basal. La formación del tubo se produce dentro de horas y los túbulos recién formadas cuantificarse fácilmente.

Ensayo de formación de tubos de células endoteliales para la<em&gt; In Vitro</em&gt; Estudio de la angiogénesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

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Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

59.0K vistas.  American University.  El ensayo de formación de tubo es un método rápido, cuantificable para medir la angiogénesis in vitro. Las células endoteliales se combinan con los medios condicionados y se sembraron en extracto de membrana basal. La formación del tubo se produce dentro de horas y los túbulos recién formadas cuantificarse fácilmente. 

Video: Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigación

Biología

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes: un modelo in vitro para el estudio de la angiogénesis (Entrevista)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Optimizado de gel de fibrina bolas de ensayo para el Estudio de la angiogénesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

A Matrigel basado en tubo de ensayo de formación para evaluar la actividad de las células tumorales vasculogénica

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Do-It-Yourself de dispositivos para la recuperación de las muestras criopreservadas de caída accidental en los tanques de almacenamiento criogénicos

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

Rata Exteriorización Mesenterio: un modelo para investigar la dinámica celulares implicados en la angiogénesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

La cuantificación de la adhesividad de la célula endotelial<em&gt; In Vitro</em

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

A Simple Bioassay for the Evaluation of Vascular Endothelial Growth Factors

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive expression of families of related receptors, a broad range of related ligands and the difficulty of dealing with primary cultures of specialized endothelial cells. Here we describe a bioassay for the detection of ligands to the vascular endothelial growth factor receptor-2 (VEGFR-2), a key transducer of signals that promote angiogenesis and lymphangiogenesis. A cDNA encoding a fusion of the extracellular (ligand-binding) region of VEGFR-2 with the transmembrane and cytoplasmic regions of the erythropoietin receptor (EpoR) is expressed in the factor-dependent cell line Ba/F3. This cell line grows in the presence of interleukin-3 (IL-3) and withdrawal of this factor results in death of the cells within 24 hr. Expression of the VEGFR-2/EpoR receptor fusion provides an alternative mechanism to promote survival and potentially proliferation of stably transfected Ba/F3 cells in the presence of a ligand capable of binding and cross-linking the extracellular portion of the fusion protein (i.e., one that can cross-link the VEGFR-2 extracellular region). The assay can be performed in two ways: a semi-quantitative approach in which small volumes of ligand and cells permit a rapid result in 24 hr, and a quantitative approach involving surrogate markers of a viable cell number. The assay is relatively easy to perform, is highly responsive to known VEGFR-2 ligands and can accommodate extracellular inhibitors of VEGFR-2 signaling such as monoclonal antibodies to the receptor or ligands, and soluble ligand traps.

We describe a simple cell-based bioassay for detecting, quantifying and monitoring the activity of members of the vascular endothelial growth factor family of ligands. The assay uses chimeric receptors expressed in a factor-dependent cell line to provide a semi-quantitative or quantitative assessment of receptor binding and cross-linking by the ligand.

Un ensayo biológico simple para la Evaluación de Factores de Crecimiento Endotelial Vascular

The analysis of receptor tyrosine kinases and their interacting ligands involved in vascular biology is often challenging due to the constitutive ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.