El trabajo es nuestro laboratorio es apoyado por becas de los Institutos Nacionales de Salud.

Preparación <ul><li> Cubreobjetos deben ser esterilizados en autoclave. </li><li> Agua destilada estéril o desionizada se debe utilizar para preparar las soluciones y cubreobjetos de lavado. </li><li> CA (40% w / v) y bis-CA (1% w / v) se esterilizan por filtración 0,2 um. Prepare un 10% de persulfato de amonio (APS, el agua 100μg/ml) poco antes de su uso y filtro estéril. Sustituir la solución APS mensuales. </li><li> Reactivos químicos como el 3-APTMS, cloroformo, glutaradehyde, NHS, y SurfaSil que no pueden ser esterilizados en autoclave se mantienen en una botella asignados utilizados únicamente para la preparación de los hidrogeles. </li><li> Para obtener mejores resultados, los hidrogeles se deben utilizar dentro de un par de días después de la incubación durante la noche con la proteína adecuada ECM. </li><li> Prepare una solución SurfaSil 10% en cloroformo (10 ml es suficiente para 20 cubreobjetos arriba) en un tubo de 50 ml de polipropileno Falcon antes de verter de hidrogel. (Nuestro laboratorio por lo general siliconizes el cubreobjetos encima durante la etapa de incubación glutaraldehído 0,5%). Agregue el cubreobjetos en el tubo Falcon y el rock por lo menos 10 min. Decantar la solución SurfaSil y secar al aire los cubreobjetos en Kimwipes en la cabina de seguridad biológica en los hidrogeles se preparará. </li><li> Prepare la solución de BSA inactivado por calor de la siguiente manera: a 20 mg / ml de solución de ácidos grasos libres BSA en PBS se incuba en un baño de agua 68 ° C durante 30 min. La solución se filtra de manera estéril y se almacenan a 4 ° C. </li></ul> Procedimiento <ol><li> Coloque una capa de Parafilm en la mitad inferior de un plato de Petri de 150 mm. </li><li> Lugar hasta 9 de 25 mm cubreobjetos en la parte superior de la Parafilm y cubrir con 1 ml de NaOH 0,1 M. Incubar durante 3 minutos y luego aspire con una línea de vacío. </li><li> Trabajo en una campana química, lugar de 0,5 ml de 3 APTMS en cada portaobjetos. Incubar durante 3 minutos y luego aspirar el APTMS. Si usted espera demasiado tiempo, una forma de espuma. </li><li> Enjuague el cubreobjetos una vez con 20 ml de agua desionizada en el mismo plato. Quitar los cubreobjetos de la cápsula, utilizando pinzas curvas y la transferencia de ellos, con sus tratos hacia arriba, a un nuevo plato de 150 mm. Lavar los cubreobjetos con agua destilada tres veces, en la mecedora, durante 10 minutos cada lavado. Si usted no puede eliminar todos los APTMS, reaccionará con el glutaraldehído en el siguiente paso y dejar un precipitado blanco turbio (Figura 1). </li><li> Descongele el glutaraldehído (~ 10 min antes de su uso). </li><li> El uso de pinzas curvas, la transferencia de los cubreobjetos de un plato limpio en capas con Parafilm y se aspira el líquido restante. Use una línea de vacío o Kimwipe para secar el agua restante, según sea necesario. </li><li> Cubra cada portaobjetos completamente con 0,5 ml de 0,5% de glutaraldehído en agua destilada estéril y se incuba durante 30 minutos en una campana química. Aspirar el glutaraldehído. Enjuague y lave el cubreobjetos como en el paso 4. Seque el cubreobjetos por completo. [Se puede parar aquí y dejar el cubreobjetos durante varias semanas en un lugar seco]. </li><li> Cuando esté listo para preparar los hidrogeles, use pinzas curvas de transferencia de los cubreobjetos, por parte reactiva, con una lámina de Parafilm que ha sido grabado en la superficie de la cabina de seguridad biológica. Asegúrese de que los cubreobjetos son planas en la superficie de Parafilm. </li><li> Prepare una solución saturada de NHS en tolueno. (Disolver una pequeña cantidad de NHS en tolueno suficiente para una experiencia particular. Continúe agregando poco a poco hasta NHS del SNS no se disuelve. La solución saturada es generalmente nublado y rosa.) </li><li> A continuación, preparar la acrilamida, bis-acrilamida, el agua y el APS para alcanzar el porcentaje deseado de acrilamida. Añadir los reactivos en tubos de microcentrífuga como se indica a continuación para un volumen total de 0.8ml. </li><li> A continuación, una alícuota a la vez, añadir el NHS y TEMED a la solución 0,8 ml de CA, vortex brevemente y vierta inmediatamente 3-5 geles con 140 l por cubreobjetos en la campana de seguridad biológica. Siempre que sea posible, todas las medidas desde este punto se debe realizar en una cabina de seguridad biológica. [Nota: cubreobjetos de diferentes tamaños pueden ser utilizados basado en las necesidades. Si de 18 mm cubreobjetos son utilizados, el uso ~ 33 l solución de CA por cubreobjetos y un cubreobjetos de 18 mm con silicona la parte superior.] <table border="1"><tbody><tr><td></td><td colspan="4"><div align="center"> Bis-AC (%) </div></td></tr><tr><td></td><td> 0,3 (rígido) </td><td> 0.15 </td><td> 0.06 </td><td> 0,03 (suave) </td></tr><tr><td></td><td> l </td><td> l </td><td> l </td><td> l </td></tr><tr><td> De agua </td><td> 402 </td><td> 522 </td><td> 594 </td><td> 618 </td></tr><tr><td> Corriente alterna </td><td> 150 </td><td> 150 </td><td> 150 </td><td> 150 </td></tr><tr><td> Bis-AC </td><td> 240 </td><td> 120 </td><td> 48 </td><td> 24 </td></tr><tr><td> APS</td><td> 8 </td><td> 8 </td><td> 8 </td><td> 8 </td></tr><tr><td> TEMED </td><td> 1 </td><td> 1 </td><td> 1 </td><td> 1 </td></tr><tr><td> NHS </td><td> 228 </td><td> 228 </td><td> 228 </td><td> 228 </td></tr></tbody></table></li><li> Rápidamente el lugar de la silicona de 25 mm cubreobjetos en la parte superior de cada gel antes de que comience a polimerizar. Además del cubreobjetos superior debe permitir que el aire acondicionado para cubrir completamente el fondo cubreobjetos. Incubar este &quot;sandwich&quot; a temperatura ambiente hasta que la AC se polimeriza. (Consulte la solución residual de CA en el tubo de microcentrífuga para determinar cuándo se ha producido la polimerización. Por lo general, unos pocos minutos para geles rígidos y un poco más de tiempo para los blandos será suficiente.). </li><li> Recoja cuidadosamente el bocadillo (guantes estériles sobre!) Y deslice la parte superior cubreobjetos hasta que sobresale por encima del gel polimerizado. A continuación, puede que saque el gel. Si usted espera demasiado tiempo antes de retirar el cubre la parte superior, el gel de rasgar el cubreobjetos se retira. </li><li> Desechar el portaobjetos superior. Coloque la parte inferior de gel cubreobjetos (en adelante, el hidrogel) en placas de 6 pocillos con 2 ml de PBS / pocillo. PBS se puede añadirse antes o después de la gel-cubreobjetos a cada pocillo. Lavar los hidrogeles con PBS tres veces, en una mecedora, a 5 min por lavado. </li><li> Repita los pasos 11-14 hasta que el número requerido de hidrogeles se han preparado. </li><li> Cubrir cada uno de hidrogel con 2 ml de una solución de fibronectina (3 mg / ml en PBS) o proteínas ECM otros (véase Klein et al. 13 para más detalles.). La proteína ECM se convierte en enlace covalente al hidrogel durante la incubación durante la noche a 4 ° C. </li><li> Aspirar la solución de ECM y el bloque del SNS no ha reaccionado con 1mg/ml inactivado por calor, los ácidos grasos libres BSA en medio libre de suero durante al menos 30 minutos a 37 ° C en un cultivo celular incubadora. Enjuague los hidrogeles una vez con PBS estéril o medio de cultivo celular. </li><li> Células de la placa en el medio de cultivo adecuado que contiene FBS. El número de células sembradas en el hidrogel debe ser determinado por el usuario en función del grado de difusión celular y la confluencia necesaria para la experimentación. Aproximadamente 10 5 células suele ser suficiente para el análisis de Western Blot y qPCR. </li><li> Tras el período de incubación, las proteínas celulares o ARNm se puede extraer. Los cubreobjetos se retira cuidadosamente de los pozos con pinzas curvas y se colocan (las células hacia abajo) en la parte superior de las gotas de 100 l de buffer de lisis que se han espaciado (2 a 3 cm) a lo largo de una lámina de Parafilm sobre la mesa del laboratorio. (Utilizamos estándar SDS muestra de amortiguación y TRIzol, respectivamente, para preparar las muestras para el Western Blot y para aislar ARN de qPCR.) Se incuban las células con el tampón de lisis de exactamente un minuto. Quitar los cubreobjetos y la transferencia de la solución amortiguadora de lisis de un tubo de microcentrífuga. Como alternativa para la extracción de ARN sólo, el usuario puede transferir cada hidrogel a una nueva de 6 pocillos y añadir 1 ml de TRIzol / bien. Incubar durante 3 minutos y retirar la solución para el almacenamiento de TRIzol en un tubo de microcentrífuga. </li></ol> Información adicional sobre los procedimientos, como la inmunofluorescencia, la tinción de BrdU, transfección, etc para que las células sembradas en hidrogeles se describe en Klein et al. 2007 13. Resultados representante Limpieza a fondo de los cubreobjetos tras la adición de APTMS es un paso importante en la producción de &quot;reactivo&quot; cubreobjetos. Si uno no puede quitar la APTMS por completo, es el que reacciona con el glutaraldehído en el paso siguiente y producir un precipitado de color blanco turbio como se ve en la figura 1A. La figura 1B muestra un cubre adecuadamente lavado y secado. Si el precipitado se desarrolla todo el proceso debe reiniciarse desde el principio como el cubreobjetos ya no es utilizable. Después de la formación del hidrogel y el recubrimiento con las proteínas ECM durante la noche, las células se pueden sembrar al día siguiente. Como muestra la Figura 2 se muestra, hay una clara diferencia entre la propagación de células en comparación con los hidrogeles rígidos blandos. Como se puede observar mediante la tinción phalloidin en fibroblastos de embriones de ratón (MEFs), las células se extendió a un mayor grado de rigidez en comparación con los hidrogeles blandos. De hecho, la mayoría de las células correspondientes a un hidrogel blando son compactas y dan menos eficiente. Aunque sólo la morfología MEF se muestra en la Figura 2, la diferencia de la propagación de células es consistente a través de varias líneas celulares de otras pruebas 11-12,14. Secretos para el Éxito <ol><li> Una vez que el procedimiento se ha iniciado, es muy importante mantener el cubreobjetos con el &quot;reactivo&quot; hacia arriba y tener en cuenta de qué lado se ha revestido. </li><li> Se deben usar guantes en todo momento durante el procedimiento para proporcionar un ambiente de trabajo que es tan estéril como sea posible. </li><li> La mayor parte de los primeros pasos se llevan a cabo entre una campana para vapores químicos y el banco de laboratorio. Todos los pasos posterioresa eliminar el exceso de glutaraldehído los cubreobjetos debe efectuarse en una cabina de seguridad biológica. </li><li> Debido a las diferencias en la propagación de células (Figura 2), se recomienda la siembra de dos veces el número de células en hidrogel blandas en comparación con los hidrogeles rígidos. </li><li> El proceso de polimerización de CA es extremadamente rápido. Para los usuarios de primera vez, se puede disminuir la cantidad de APS o TEMED en la solución para prolongar el proceso de polimerización. No trate de preparar más de un cubreobjetos de unos pocos a la vez. </li><li> En los estudios de ciclo de la célula donde se requiere sincronización en G0, por lo general el suero de hambre las células en placas de cultivo de plástico, trypsinize las células y resembrar en los hidrogeles de cumplimientos diferentes, en presencia de mitógenos (FBS y / o factores de crecimiento). Este procedimiento asegura que la población a partir de G0-sincronizado células es idéntico en todas las muestras. Sin embargo, para muchos estudios de transducción de señales, el período de estimulación por mitógenos requerida no puede ser lo suficientemente largo para permitir que el apego y la propagación de las células. En este caso, el suero de hambre las células de los hidrogeles y luego directamente a estimular con mitógenos. </li></ol><img src="/files/ftp_upload/2089/2089fig1a.jpg" alt="Figura 1a" /> Figura 1A. Inapropiadamente lavados cubreobjetos. Tras la adición de APTMS, cubreobjetos se lavó durante 1-2 minutos antes de la adición de la solución de glutaraldehído. El precipitado se forma en el cubreobjetos que no haya sido lavado de acuerdo a los pasos descritos en el procedimiento. <img src="/files/ftp_upload/2089/2089fig1b.jpg" alt="Figura 1b" /> Figura 1B. Lavados cubreobjetos. Además Después de APTMS, cubreobjetos se lavó tres veces 10 minutos cada una antes de la adición de la solución de glutaraldehído. No precipitado forma en el cubreobjetos. <img src="/files/ftp_upload/2089/2089fig2.jpg" alt="Figura 2" /> Figura 2. Morfología de las células en los hidrogeles de diferente rigidez. Establecida MEFs fueron sembradas en recubiertos de fibronectina hidrogeles de la rigidez de alta o baja durante 9 horas. Tras el período de incubación, las células fueron fijadas, permeabilized y teñidas con FITC-faloidina que se une a la F-actina. MEFs en geles de alta rigidez presentan las fibras de estrés y se extiende mucho en comparación con las sembradas en hidrogeles baja rigidez. Barra de escala = 50 micras. <img src="/files/ftp_upload/2089/2089fig3.jpg" alt="Figura 3" /> Figura 3. Representante resultado de la PCR cuantitativa de los niveles de ARNm de la ciclina D1 ratón. Suero fibroblastos embrionarios de ratón muerto de hambre fueron chapados en alto (3% de acrilamida) o bajo (0,3% de acrilamida) hidrogeles rigidez y estimulados con FBS al 10% durante 9 horas. Tras la extracción de ARN, en tiempo real, análisis de PCR cuantitativa se realizó para los niveles de ARNm de la ciclina D1 (normalizado a 18S ARN). G0 representa ciclina D1 ARNm de las células en reposo. Niveles de ciclina D1 mRNA de aumentar de forma significativa en los hidrogeles rígidos, pero no en los hidrogeles blandos. Los datos son la media + / - SD de duplicar las reacciones de PCR.

<ol>
	<li>Beattie, D., Xu, C., Vito, R., Glagov, S., Whang, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+analysis+of+heterogeneous,+atherosclerotic+human+aorta.">Mechanical analysis of heterogeneous, atherosclerotic human aorta.</a> J Biomech Eng. 120, 602-607 (1998).</li><li>Bernini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness,+intima-media+thickness+and+carotid+artery+fibrosis+in+patients+with+primary+aldosteronism.">Arterial stiffness, intima-media thickness and carotid artery fibrosis in patients with primary aldosteronism.</a> J Hypertens. 26, 2399-2405 (2008).</li><li>Boonyasirinant, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+stiffness+is+increased+in+hypertrophic+cardiomyopathy+with+myocardial+fibrosis:+novel+insights+in+vascular+function+from+magnetic+resonance+imaging.">Aortic stiffness is increased in hypertrophic cardiomyopathy with myocardial fibrosis: novel insights in vascular function from magnetic resonance imaging.</a> J Am Coll Cardiol. 54, 255-2562 (2009).</li><li>Discher, D. E., Janmey, P., Wang, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cells+feel+and+respond+to+the+stiffness+of+their+substrate.">Tissue cells feel and respond to the stiffness of their substrate.</a> Science. 310, 1139-1143 (2005).</li><li>Duprez, D. A., Cohn, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+stiffness+as+a+risk+factor+for+coronary+atherosclerosis.">Arterial stiffness as a risk factor for coronary atherosclerosis.</a> Curr Atheroscler Rep. 9, 139-144 (2007).</li><li>Lee, R. 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Un elemento crucial del proceso de polimerización de hidrogel es para evitar la formación de burbujas de aire que permiten que las células se unen a la cubreobjetos de vidrio en lugar de que el hidrogel ECM-revestidos en sí. Esto se puede evitar con cuidado pipeteando la solución de polimerización después de agitación y visual asegurándose que no queden burbujas de aire han quedado atrapados en el gel. Nosotros siempre recomendamos la preparación adicional &quot;reactivo&quot; cubreobjetos y los hidrogeles par...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Glutaraldehyde, 70%</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>G7776</td>
<td>Store at -20&deg;C</td>
</tr>
<tr>
<td>3-APTMS (3-Aminopropyltrimethosysilane 97%)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>SurfaSil Siliconizing Fluid</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>42800</td>
<td>Store at room temperature</td>
</tr>
<tr>
<td>NHS (N-hydroxysucinimide Ester)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A-8060</td>
<td>Store at 4&deg;C Replace monthly</td>
</tr>
<tr>
<td>Albumin, bovine serum, essentially fatty acid free</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>A6003-100G</td>
<td>Store at 4&deg;C</td>
</tr>
<tr>
<td>Coverslips (25mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-86 25 Cir 1D</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips (18mm)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-545-84 18 Cir 1D</td>
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studying the effects of matrix stiffness on cellular function using acrylamide-based hydrogels

La rigidez del tejido es un factor determinante de la función celular, y cambios en la rigidez del tejido son comúnmente asociados con la fibrosis, cáncer y enfermedades cardiovasculares 1-11. Los enfoques tradicionales de biología celular al estudio de la función celular implica el cultivo de células sobre un sustrato rígido (platos de plástico o de vidrio cubreobjetos), que no puede explicar el efecto de una ECM elástica o las variaciones en la rigidez de ECM entre los tejidos. Para crear un modelo in vivo las condiciones del tejido cumplimiento in vitro, y otros utilizan ECM-revestidos hidrogeles. En nuestro laboratorio, los hidrogeles se basan en la poliacrilamida que puede imitar la serie de incumplimientos del tejido biológico visto 12. &quot;Reactiva&quot; cubreobjetos son generados por la incubación con NaOH seguido por la adición de 3-APTMS. El glutaraldehído se utiliza para reticular el 3-APTMS y el gel de poliacrilamida. Una solución de acrilamida (AC), bis-acrilamida (Bis-AC) y persulfato de amonio se utiliza para la polimerización del hidrogel. N-hidroxisuccinimida (NHS) se incorpora a la solución de CA para reticular las proteínas ECM al hidrogel. Después de la polimerización del hidrogel, la superficie del gel está recubierto con una proteína de elección ECM como la fibronectina, vitronectina, colágeno, etc La rigidez de un hidrogel puede ser determinada por la reología o microscopía de fuerza atómica (AFM) y ajustado variando el porcentaje de AC y / o AC-bis en la solución 12. De esta manera, la rigidez sustrato se puede adaptar a la rigidez de los tejidos biológicos, que también puede ser cuantificado usando la reología o AFM. Células pueden ser sembradas en estos hidrogeles y cultivadas según las condiciones experimentales necesarias. Imágenes de las células y su recuperación para el análisis molecular es sencillo. Para este artículo, se define sustratos blandos como los que tienen módulos de elasticidad (E) &lt;3000 Pascal y sustratos rígidos / tejidos como aquellos con E&gt; 20.000 Pascal.

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Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

El efecto de la rigidez del sustrato en la función celular puede ser modelada<em&gt; In vitro</em&gt; Utilizando los hidrogeles de poliacrilamida de diferentes incumplimientos.

El estudio de los efectos de la matriz de rigidez en la función celular usando como base la acrilamida-hidrogeles

Tissue stiffness is an important determinant of cellular function, and changes in tissue stiffness are commonly associated with fibrosis, cancer and ...

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Biology

21.9K vistas.  University of Pennsylvania. El efecto de la rigidez del sustrato en la función celular puede ser modelada In vitro Utilizando los hidrogeles de poliacrilamida de diferentes incumplimientos.

Video: Studying the Effects of Matrix Stiffness on Cellular Function using Acrylamide-based Hydrogels

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

La encapsulación celular en hidrogeles 3D para ingeniería de tejidos

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Investigación

Biología

Measuring the Bending Stiffness of Bacterial Cells Using an Optical Trap

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic three-dimensional spring made of light that is formed when a high-intensity laser beam is focused to a very small spot by a microscope's objective lens. To bend a cell, we first bind live bacteria to a chemically-treated coverslip. As these cells grow, the middle of the cells remains bound to the coverslip but the growing ends are free of this restraint. By inducing filamentous growth with the drug cephalexin, we are able to identify cells in which one end of the cell was stuck to the surface while the other end remained unattached and susceptible to bending forces. A bending force is then applied with an optical trap by binding a polylysine-coated bead to the tip of a growing cell. Both the force and the displacement of the bead are recorded and the bending stiffness of the cell is the slope of this relationship.

We present a protocol for bending filamentous bacterial cells attached to a cover-slip surface with an optical trap to measure the cellular bending stiffness.

La medición de la resistencia a la flexión de las células bacterianas usando una trampa óptica

We developed a protocol to measure the bending rigidity of filamentous rod-shaped bacteria. Forces are applied with an optical trap, a microscopic ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

La identificación de los efectos de las mutaciones BRCA1 en recombinación homóloga utilizando células que expresan endógeno de tipo salvaje BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

El análisis de la función de las pequeñas GTPasas de la microinyección de plásmidos en células epiteliales polarizadas

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase (COX/SDH) Double-labeling Histochemistry

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of aging suggests a role for mtDNA mutations, which can alter bioenergetics homeostasis and cellular function, in the aging process 3. A wealth of evidence has been compiled in support of this theory 1,4, an example being the mtDNA mutator mouse 5; however, the precise role of mtDNA damage in aging is not entirely understood 6,7.
Observing the activity of respiratory enzymes is a straightforward approach for investigating mitochondrial dysfunction. Complex IV, or cytochrome c oxidase (COX), is essential for mitochondrial function. The catalytic subunits of COX are encoded by mtDNA and are essential for assembly of the complex (Figure 1). Thus, proper synthesis and function are largely based on mtDNA integrity 2. Although other respiratory complexes could be investigated, Complexes IV and II are the most amenable to histochemical examination 8,9. Complex II, or succinate dehydrogenase (SDH), is entirely encoded by nuclear DNA (Figure 1), and its activity is typically not affected by impaired mtDNA, although an increase might indicate mitochondrial biogenesis 10-12. The impaired mtDNA observed in mitochondrial diseases, aging, and age-related diseases often leads to the presence of cells with low or absent COX activity 2,12-14. Although COX and SDH activities can be investigated individually, the sequential double-labeling method 15,16 has proved to be advantageous in locating cells with mitochondrial dysfunction 12,17-21.
Many of the optimal constitutions of the assay have been determined, such as substrate concentration, electron acceptors/donors, intermediate electron carriers, influence of pH, and reaction time 9,22,23. 3,3'-diaminobenzidine (DAB) is an effective and reliable electron donor 22. In cells with functioning COX, the brown indamine polymer product will localize in mitochondrial cristae and saturate cells 22. Those cells with dysfunctional COX will therefore not be saturated by the DAB product, allowing for the visualization of SDH activity by reduction of nitroblue tetrazolium (NBT), an electron acceptor, to a blue formazan end product 9,24. Cytochrome c and sodium succinate substrates are added to normalize endogenous levels between control and diseased/mutant tissues 9. Catalase is added as a precaution to avoid possible contaminating reactions from peroxidase activity 9,22. Phenazine methosulfate (PMS), an intermediate electron carrier, is used in conjunction with sodium azide, a respiratory chain inhibitor, to increase the formation of the final reaction products 9,25. Despite this information, some critical details affecting the result of this seemly straightforward assay, in addition to specificity controls and advances in the technique, have not yet been presented.

Visualization of Mitochondrial Respiratory Function using Cytochrome C Oxidase / Succinate Dehydrogenase COX/SDH Double-labeling Histochemistry

The cytochrome c oxidase/sodium dehydrogenase (COX/SDH) double-labeling method allows for direct visualization of mitochondrial respiratory enzyme deficiencies in fresh-frozen tissue sections. This is a straightforward histochemical technique and is useful in investigating mitochondrial diseases, aging, and aging-related disorders.

La visualización de la función respiratoria mitocondrial utilizando citocromo C Oxidasa / Succinato Deshidrogenasa (COX / SDH) de doble etiquetado Histoquímica

Mitochondrial DNA (mtDNA) defects are an important cause of disease and may underlie aging and aging-related alterations 1,2. The mitochondrial theory of ...

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for "less than normal O2". Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition &lt;10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.

Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans

This paper details how to use continuous-flow hypoxia chambers to generate atmospheres with defined concentrations of O2 to understand biological responses to decreased O2. This system is easy to setup and maintain, and flexible enough to suit a wide range of O2 concentrations and model systems

Creación de ambientes definidos de gases para el Estudio de los efectos de la hipoxia en el<em&gt; C. elegans</em

Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

Medición de los efectos de las bacterias en<em&gt; C. Elegans</em&gt; Comportamiento El uso de un ensayo de retención de huevo

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Analysis of Beta-cell Function Using Single-cell Resolution Calcium Imaging in Zebrafish Islets

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to hyperglycemia and severe, life-threatening consequences. Understanding how the beta-cells operate under physiological conditions and what genetic and environmental factors might cause their dysfunction could lead to better treatment options for diabetic patients. The ability to measure calcium levels in beta-cells serves as an important indicator of beta-cell function, as the influx of calcium ions triggers insulin release. Here we describe a protocol for monitoring the glucose-stimulated calcium influx in zebrafish beta-cells by using GCaMP6s, a genetically encoded sensor of calcium. The method allows monitoring the intracellular calcium dynamics with single-cell resolution in ex vivo mounted islets. The glucose-responsiveness of beta-cells within the same islet can be captured simultaneously under different glucose concentrations, which suggests the presence of functional heterogeneity among zebrafish beta-cells. Furthermore, the technique provides high temporal and spatial resolution, which reveals the oscillatory nature of the calcium influx upon glucose stimulation. Our approach opens the doors to use the zebrafish as a model to investigate the contribution of genetic and environmental factors to beta-cell function and dysfunction.

Beta-cell functionality is important for blood-glucose homeostasis, which is evaluated at single-cell resolution using a genetically encoded reporter for calcium influx.

Análisis de la función de célula Beta usando proyección de imagen de resolución sola célula calcio en islotes de pez cebra

Pancreatic beta-cells respond to increasing blood glucose concentrations by secreting the hormone insulin. The dysfunction of beta-cells leads to ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Un modelo de cultivo celular de adipocitos para estudiar el impacto de la modulación de proteínas y microARN en la función de los adipocitos

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Análisis de la función E3 Ubiquitina Ligasa

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...