Nos gustaría dar las gracias a Philippa Warren por su amable ayuda.

1. Límite del ensayo: <ol><li> Preparación básica: la cámara de diapositivas están recubiertas con poli-D-lisina portaobjetos de vidrio durante la noche y estériles se preparan antes de comenzar el experimento. Medio se prepara con Dulbecco Modificado Medio Eagle (DMEM), suplementado con 10% de suero fetal bovino (FCS), y el 1% penicilina-estreptomicina-Fungizon (PSF). También una botella de libre Calcio / Magnesio solución salina balanceada de Hanks (HBSS) se prepara. </li><li> Primaria las células de Schwann de rata en cultivo en frasco se trypsinized durante 3 minutos con 0,1% de tripsina. Cultivos primarios de astrocitos de rata se trypsinized durante 5 minutos con 0,1% de tripsina. La tripsina es inactivado mediante la adición de DMEM suplementado con 10% de FCS y 1% de PSF. </li><li> Trypsinized células de Schwann y astrocitos se transfieren a separar 15 ml tubos falcon y se centrifuga a 300 G durante 5 minutos. </li><li> Las células de Schwann y astrocitos están nuevamente en suspensión en la densidad de 2 x 10 6 (ver sección de resultados representativos de las variaciones en la densidad celular). </li><li> 50 l gota de suspensión de células de Schwann se coloca en un extremo del PDL pozos revestidos cámara de diapositivas. Una tira de cristal que se utiliza para desprestigiar a la caída hacia el centro de la cámara para generar un borde recto. (Usando un cortador de vidrio, un cubreobjetos de vidrio rectangular se cortan longitudinalmente, de manera que se ajuste al ancho de la cámara de diapositivas utilizadas para los ensayos de límite) </li><li> Una segunda caída de 50 l de la suspensión de los astrocitos se colocó en el extremo opuesto de la misma y se unta bien hacia el centro para crear un borde recto paralelo al desprestigio primera caída con una pequeña diferencia de 0,2 mm entre ellos. </li><li> La cámara de diapositivas que contienen las gotas se colocan en la incubadora a 37 ° C durante 2 horas. </li><li> Después de 2 horas, los cultivos se lavaron con DMEM para eliminar no inscritos los desechos celulares y un nuevo soporte de DMEM / FCS al 10% / 1% de fibras discontinuas de poliéster añadió. Nota: DMEM / FCS al 10% / 1% de fibras discontinuas de poliéster se puede complementar con forskolina (2μM) y el extracto de pituitaria bovina (BPE) (10μg / ml) estimulan la proliferación de células de Schwann. BPE suministrará los factores de crecimiento necesarios para permitir a largo plazo la cultura de células de Schwann y la forskolina eleva los niveles de adenosina cíclico monofosfato en las células de Schwann para inducir la proliferación </li><li> Las células se cultivan durante unos 80-10 días a 37 º C, el 7% de CO 2 incubadora. El medio necesita ser cambiada cada 3 días. Los dos frentes de células con el tiempo llegará a los demás y un límite claro se forma entre los dos tipos de células. En esta etapa, los experimentos pueden llevarse a cabo el análisis de los factores que intervienen en la formación de frontera. </li><li> Co-cultivo pueden ser fijas con formaldehído al 4% y las células de Schwann se immunostained con anticuerpos anti-P75 y astrocitos con anti-GFAP anticuerpos para identificar las células en co-cultivo. P75 anticuerpo reconoce receptor de baja afinidad de NGF en células de Schwann que los astrocitos falta. Anti-GFAP anticuerpo reconoce proteína glial firbrillary ácido que se localiza en el citoplasma de astrocitos. (Por favor vea la Figura 1A de frontera representante) </li></ol> 2. La migración del ensayo: <ol><li> 4 placas (15 mm de pozos) se PDL cubiertas durante la noche y se preparó para la creación de monocapas de astrocitos. </li><li> La cultura de astrocitos primarios se trypsinized durante 5 minutos con 0,1% de tripsina. Tripsina se inactiva mediante la adición de DMEM suplementado con 10% de FCS y 1% de fibras discontinuas de poliéster y celular se centrifuga a 300 G durante 5 minutos. (Ver Figura 2) </li><li> Los astrocitos son re-suspendido en el 1x10 5 células / ml. 1 ml de esta solución se añade a cada uno sensibilizados PDL y en 4 placas. Los astrocitos son cultivados durante 24-48 horas hasta que la monocapa es totalmente confluente. </li><li> Para evaluar la migración de células de Schwann en monocapas de astrocitos, en paralelo a la creación de monocapas de astrocitos, circular PDL-cubreobjetos de vidrio recubierto se coloca en un tubo de 50 ml y aplastó con una pipeta de plástico para crear pequeños fragmentos de cristal. </li><li> Fragmentos de vidrio se transfieren a un pozo de seis platos y placa y el uso de fórceps adecuado fragmentos de tamaño se seleccionan y se colocan en otros pozos. 5 fragmentos de vidrio se colocan en cada pocillo en placas de 6 pocillos. </li><li> Primaria las células de Schwann se cultivan en frasco se trypsinized durante 3 minutos con 0,1% de tripsina. Trypsinized células de Schwann se transfieren a separar 15 ml tubo Falcon y se centrifuga a 300 G durante 5 minutos. Las células de Schwann se resuspendieron a 2 x 10 6 células / ml en DMEM/10% FCS / 1% de PSF. </li><li> 20 gotas de L de la suspensión células de Schwann se añade en cada cinco fragmentos cubreobjetos colocado en placas de 6 pocillos y las células se incuban a 37 ° C CO, 7% 2 durante 2 horas. </li><li> Después de 2 horas, las células de Schwann se unen a los fragmentos cubreobjetos y DMEM/10% FCS / 1% de fibras discontinuas de poliéster (Complementado con 2μM de forskolina y 10μg / ml de BPE se añade a cada pocillo de modo que los fragmentos están completamente cubiertos con el medio) . </li><li> El 6 y placas que contienen los fragmentos se colocan en la incubadora durante 24-48 horas hasta que los fragmentos de vidrio son totalmente Confluida con las células de Schwann. </li><li> Una vez que los fragmentos son confluentes con células de Schwann, cada fragmento se recoge con cuidado el uso de fórceps fuerte y se sumerge en HBSS para eliminar las células sueltas y luego la cara invertida hacia abajo sobre monocapa de astrocitos. </li><li> Cada pozo se cubre con 1 ml de DMEM/10% FCS / 1% de las células de Schwann y fibras discontinuas de poliéster se les permita emigrar de 24 a 48 periodo. Durante este período los reactivos experimentales, tales como factores de crecimiento se puede agregar a evaluar su efecto sobre el número y la distancia de la migración de las células de Schwann. </li><li> Tras el período de migración, los fragmentos de vidrio se fijan en la parte inferior de cada pozo mediante la adición de 4% de paraformaldehído (PFA) durante 30 minutos. Después de la fijación, las células de Schwann inmunotinción para p75 (el reconocimiento de baja afinidad del receptor de NGF) y luego mancha ABC strepavidin kit-diaminobencidina (DAB), para permitir la visualización de las células de Schwann bajo microscopio de luz. </li></ol> 3. Los resultados representativos: Después de 8-10 días de Schwann, astrocitos co-cultivos muestran un límite claro entre los dos tipos de células. Este límite es más pronunciado si forskolin y BPE se utiliza en la cultura como la proliferación de células de Schwann se ha mejorado. Si no hay ningún límite se observa durante el período de 10 días, el tiempo de la cultura se puede aumentar aún más hasta los límites establecidos. Cambio de la relación de la densidad celular es una forma alternativa para aumentar la formación de límites. Una proporción de 3:1 de Schwann: densidad celular de astrocitos se pueden utilizar en la preparación de suspensiones de células 8,18. Después de la inmunotinción para identificar las células de Schwann y astrocitos, células para evaluar una línea de mezcla se pueden sacar en donde el límite está establecido y el número de la migración de las células de Schwann en el territorio de astrocitos se pueden contar bajo el microscopio de fluorescencia. A continuación se muestran dos ejemplos de imágenes obtenidas fronteras, con una bien formada la segregación de las células de Schwann y astrocitos (Figura 1) y una en la que las células no se han separado y en diferentes territorios, debido a una mala técnica (fig. 1B). <img src="/files/ftp_upload/2214/2214fig2.jpg" alt="Figura 1" /> . Figura 1 Límites del ensayo: Schwann, astrocitos interacción. A) con 10 días de co-cultivo de ensayo en presencia de forskolina y BPE para estimular la proliferación de células de Schwann demuestra una frontera clara entre los dos tipos de células (rojo = P75, verde = GFAP). B) A 10 días de co-cultivo de ensayo donde la frontera no se ha formado y los dos frentes de células mixtas. Esto se debió a la mezcla de las dos gotas, mientras que la colocación de las gotas de células de Schwann y astrocitos uno junto al otro durante la preparación de la frontera (rojo = P75, verde = GFAP). <img src="/files/ftp_upload/2214/2214fig1.jpg" alt="Figura 2" /> Figura 2. Imagen representativa de la monocapa de astrocitos confluentes Los ensayos de migración evaluar el movimiento de un tipo de células en la superficie de otro tipo de células y por lo tanto evaluar un fenómeno diferente de la de la formación de frontera. La práctica es necesaria para asegurar la inversión del cubreobjetos en monocapas no causar daños a la célula de Schwann, cubierta de fragmentos de vidrio y monocapas de astrocitos con las pinzas afiladas. Para evaluar la migración de las células de Schwann, la distancia máxima de la migración y el número de células que migran desde el borde del cubreobjetos se puede medir bajo microscopio de luz. A continuación se muestra la imagen de las células de Schwann se tiñeron con anticuerpos P75 mediante la tinción de DAB en la superficie de monocapa de astrocitos (Figura 3). <img src="/files/ftp_upload/2214/2214fig3.jpg" alt="Figura 3" /> Figura 3. Migración de ensayo utilizando el ensayo cubreobjetos invertido. A) las células de Schwann (marrón, immunostained con anticuerpos p75) migrando desde el borde del cubreobjetos. La flecha apunta a la dirección de la migración de las células fuera del cubreobjetos.

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+ensheathing+cells+induce+less+host+astrocyte+response+and+chondroitin+sulphate+proteoglycan+expression+than+Schwann+cells+following+transplantation+into+adult+CNS+white+matter.">Olfactory ensheathing cells induce less host astrocyte response and chondroitin sulphate proteoglycan expression than Schwann cells following transplantation into adult CNS white matter.</a> Exp Neurol. 184, 237-246 (2003).</li><li>Ghirnikar, R. S., Eng, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte-Schwann+cell+interactions+in+culture.">Astrocyte-Schwann cell interactions in culture.</a> Glia. 11, 367-377 (1994).</li><li>Ghirnikar, R. S., Eng, L. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrogliosis+limits+the+integration+of+peripheral+nerve+grafts+into+the+spinal+cord.">Astrogliosis limits the integration of peripheral nerve grafts into the spinal cord.</a> Brain Res. 277, 175-180 (1983).</li><li>Fok-Seang, J., Mathews, G. A., ffrench-Constant, C., Trotter, J., Fawcett, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+oligodendrocyte+precursors+on+astrocytes+and+meningeal+cells.">Migration of oligodendrocyte precursors on astrocytes and meningeal cells.</a> Dev Biol. 171, 1-15 (1995).</li><li>Wilby, M. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+migration+is+integrin-dependent+and+inhibited+by+astrocyte-produced.">Schwann cell migration is integrin-dependent and inhibited by astrocyte-produced.</a> Glia. , (2010).</li><li>Santos-Silva, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF/heparin+differentially+regulates+Schwann+cell+and+olfactory+ensheathing+cell+interactions+with+astrocytes:+a+role+in+astrocytosis.">FGF/heparin differentially regulates Schwann cell and olfactory ensheathing cell interactions with astrocytes: a role in astrocytosis.</a> J Neurosci. 27, 7154-7167 (2007).</li><li>Afshari, F. T., Kwok, J. C., Fawcett, J. 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Los ensayos descritos anteriormente se han utilizado en diversos estudios que demuestran el papel de los múltiples factores que intervienen en la formación de frontera entre los Schwann, astrocitos y células de Schwann migración limitada en el entorno de los astrocitos. Comprensión de los mecanismos que subyacen a estos eventos es fundamental, ya que permitiría el desarrollo de estrategias para optimizar y mejorar la integración de los injertos de células de Schwann después del tras...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>41966-029</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-088</td>
<td>&nbsp;</td>
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10091-148</td>
<td>&nbsp;</td>
<tr>
<td>PSF</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15240-062</td>
<td>&nbsp;</td>
<tr>
<td>BPE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>13028-014</td>
<td>&nbsp;</td>
<tr>
<td>Forskolin</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>344273</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips</td>
<td>&nbsp;</td>
<td>VWR-International</td>
<td>631-0149</td>
<td>&nbsp;</td>
<tr>
<td>Coverlips(rectangle)</td>
<td>&nbsp;</td>
<td>MENZEL-GLASER (Supplied by Thermo Scientifics)</td>
<td>BB022050A1</td>
<td>&nbsp;</td>
<tr>
<td>Chamber slides</td>
<td>&nbsp;</td>
<td>Nunc-LabTek </td>
<td>177380</td>
<td>&nbsp;</td>
<tr>
<td>4 well plates, 6 well plates</td>
<td>&nbsp;</td>
<td>Nunc</td>
<td>&nbsp;</td>
<td>4 well plates 
6 well plates</td>
</tr>
<tr>
<td>rat p75 antibody</td>
<td>&nbsp;</td>
<td>Millipore, Temecula,California</td>
<td>MAB365</td>
<td>&nbsp;</td>
<tr>
<td>GFAP antibody</td>
<td>&nbsp;</td>
<td>Dako</td>
<td>Z0334</td>
<td>&nbsp;</td>
<tr>
<td>Secondary antibodies (Alexa-conjugated)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A-11004 A-11034</td>
<td>Goat anti mouse 568 
Goat anti Rabbit 488</td>
</tr>
<tr>
<td>Secondary biotinylated </td>
<td>&nbsp;</td>
<td>Vector</td>
<td>&nbsp;</td>
<td>Goat anti mouse</td>
</tr>
<tr>
<td>DAB tablets </td>
<td>&nbsp;</td>
<td>Sigma-aldrich</td>
<td>D4418</td>
<td>&nbsp;</td>
<tr>
<td>ABC elite kit</td>
<td>&nbsp;</td>
<td>Vector</td>
<td>PK-6100</td>
<td>&nbsp;</td>
<tr>
<td>Fine Forceps </td>
<td>&nbsp;</td>
<td>FST</td>
<td>11295-10</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P6168</td>
<td>&nbsp;</td>
<tr>
<td>Fluorosave</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>345789</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

analysis of schwann-astrocyte interactions using in vitro assays

Las células de Schwann son una de las celdas habitualmente utilizadas en las estrategias de reparación después de lesiones de la médula espinal. Las células de Schwann son capaces de soportar la regeneración axonal y el brote de crecimiento secretan factores de crecimiento de 1,2 y ofrecer la promoción de tres moléculas de adhesión y moléculas de la matriz extracelular 4. Además se mielinizar los axones mielina en el sitio de la lesión 5. Sin embargo después del trasplante, las células de Schwann no migran desde el sitio de implante y no se mezclan con los astrocitos de acogida 6,7. Esto se traduce en la formación de una frontera clara entre las células de Schwann y astrocitos, creando un obstáculo para los axones en crecimiento tratando de salir de la parte posterior del injerto en el tejido del huésped proximal y distal. Los astrocitos en contacto con las células de Schwann también se someten a la hipertrofia y un máximo de regular las moléculas inhibidoras 08.13. En ensayos in vitro se han utilizado para modelar la célula de Schwann, astrocitos y las interacciones han sido importantes en la comprensión de los mecanismos subyacentes al comportamiento celular. Estos ensayos in vitro son los límites del ensayo, donde se hizo un co-cultivo con dos células diferentes con cada tipo de célula que ocupan territorios diferentes con sólo una pequeña brecha que separa los dos frentes de la célula. Como las células se dividen y emigran, los dos frentes celular se acercan el uno al otro y finalmente colisionan. Esto permite que el comportamiento de las dos poblaciones celulares que se analizan en la frontera. Otra variante de la misma técnica que consiste en mezclar las dos poblaciones celulares en la cultura y con el tiempo los dos tipos de células segregan con las células de Schwann se agruparon como islas en medio de los astrocitos en conjunto la creación de múltiples Schwann, astrocitos límites. El segundo ensayo utilizados en el estudio de la interacción de dos tipos de células es el ensayo de la migración, donde puede ser rastreado movimiento celular en la superficie del tipo de células en monocapa 14,15. Este ensayo se conoce comúnmente como ensayo cubreobjetos invertido. Las células de Schwann se cultivan en pequeños fragmentos de vidrio y se invierten boca abajo sobre la superficie de las monocapas de astrocitos y la migración se evalúa desde el borde del cubreobjetos. Ambos ensayos han sido fundamentales en el estudio de los mecanismos subyacentes que implica la exclusión celular y la formación de frontera. Algunas de las moléculas identificadas mediante estas técnicas, como las N-cadherinas 15, condroitina sulfato proteoglicanos (CSPGs) 16,17, FGF / Heparina 18, Efe / Efrinas 19. En este artículo se pretende describir ensayo límite y ensayo de migración en forma gradual y aclarar los posibles problemas técnicos que pudieran ocurrir.

Watch this Scientific Journal Video about Analysis of Schwann-astrocyte Interactions Using In Vitro Assays at JoVE.com

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

En este artículo se pretende describir en forma gradual el uso común<em&gt; In vitro</em&gt; Métodos de evaluación utilizados en el estudio de la célula de Schwann-asrtocyte interacciones.

El análisis de Schwann-astrocito interacciones utilizando In Vitro Ensayos

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

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Research

JoVE Journal

Biology

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

12.1K vistas.  University of Cambridge. En este artículo se pretende describir en forma gradual el uso común In vitro Métodos de evaluación utilizados en el estudio de la célula de Schwann-asrtocyte interacciones.

Video: Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

Detección rápida homogénea de Ensayos Biológicos Usando el Sistema de modulación magnética biosensores

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

Investigación

Biología

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

Análisis in vitro de PDZ-dependientes de complejos macromoleculares de señalización CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

En la migración celular y la invasión ensayos in vitro

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

Análisis de las reacciones de procesamiento del ARN que utilizan teléfonos Free Systems: 3 &#39;End división de pre-mRNA Sustratos<em&gt; In vitro</em

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of hyperphosphorylated tau protein in the form of neurofibrillary tangles (NFTs). The density of NFT correlates well with cognitive impairment and other neurodegenerative symptoms, thus prompting the endeavor of developing tau aggregation-based therapeutics. Thus far, however, tau aggregation assays use recombinant or synthetic tau that is devoid of the pathology-related phosphorylation marks. Here we describe two assays using recombinant, hyperphosphorylated tau as the subject. These assays can be scaled up for high-throughput screens for compounds that can modulate the kinetics or stability of hyperphosphorylated tau aggregates. Novel therapeutics for Alzheimer's disease and other tauopathies can potentially be discovered using hyperphosphorylated tau isoforms.

In Vitro Aggregation Assays Using Hyperphosphorylated Tau Protein

Unmodified and hyperphosphorylated tau proteins were used in two in vitro aggregation assays to reveal the hyperphosphorylation-dependent fast aggregation kinetics. These assays pave the way for future screens for compounds that can modulate the propensity of hyperphosphorylated tau to form fibrils that underlie the progression of Alzheimer&#8217;s disease.

En Vitro agregación ensayos usando hiperfosforilada proteína tau

Alzheimer's disease is one of a large group of neurodegenerative disorders known as tauopathies that are manifested by the neuronal deposits of ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Análisis comparativo de la hormona de crecimiento humano en suero utilizando SPRI, Nano-SPRI y ELISA Ensayos

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Protocolo de laboratorio para análisis genéticos de contenido intestinal de macroinvertebrados acuáticos con grupo-específica rDNA cartillas

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Efecto de proteínas fluorescentes de fusión socios en ensayos de toxicidad de poliglutaminas de levadura

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Análisis de microbiota utilizando PCR de dos pasos y secuenciación genética de rRNA 16S de próxima generación

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

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 ...