La investigación fue apoyada por subvenciones del NIH EY012031 y EY0182175 y la Fundación FM Kirby.

Las pinzas ópticas para la manipulación de células aisladas en la cultura Las fuerzas de la captura de las pinzas ópticas se generan a partir del impulso de la luz (Ashkin, 1991;. Ashkin et al, 1986). A pesar de estas fuerzas fácil atrapar las células en suspensión, que no son capaces de mover las células que se adhieren a la superficie. Por lo tanto, a reducir la adherencia de la placa de cultivo en el punto donde las células están atrapados, los cubreobjetos está recubierta con poli-2-hidroxietilo (poli-HEMA) (Sigma Chemical Co., St Louis, MO), un compuesto no tóxico con la célula de repelente propiedades empleadas previamente para estudios de adherencia en las células endoteliales (Folkman y Moscona, 1978). Mientras que el poli-HEMA cubre la mitad de la placa de cultivo, la otra mitad está cubierta con un sustrato que soporta el crecimiento celular. En nuestro laboratorio, Sal-1 se utiliza como sustrato para el cultivo de células de la retina salamandra. El procedimiento para la fabricación de Sal-1 y poli-HEMA platos recubiertos se describe a continuación: Fabricación de placas de cultivo <ol><li> Cubres limpio (# 1 VWR Scientific Inc., Media, PA) en ácido. Esto cubre objetos fue seleccionada por su grosor de tan sólo 0,1 mm, necesaria para los objetivos de gran apertura numérica necesario para enfocar el láser. </li><li> Para crear un área de repelente de la célula, se disuelven 20 mg de poli-HEMA (Sigma Chemical Co., St Louis, MO) en 1 ml de etanol al 95% durante la noche a temperatura ambiente. Preferiblemente, esta solución debe utilizarse dentro de un mes de preparación. </li><li> Apuntalar la cubres en una posición casi vertical en un ambiente libre de polvo, como una campana. </li><li> Coloque una o dos gotas de solución de poli-HEMA en la parte superior de la cubre objetos desde la punta de plástico de una micropipeta 200μl. Las gotas (50-100μl cada uno) debería limitarse a la mitad de los cubre-objetos. La pendiente pronunciada se asegura que el líquido fluye por la superficie del cubre objetos en el fondo, proporcionando una capa delgada y uniforme de poli-HEMA. Deje que la cubres recubiertos de poli-HEMA a secar en la campana durante 30-60 minutos a temperatura ambiente. </li><li> Marque el lado de la poli-HEMA el cubre con una pluma de punta fina cerca del borde. </li><li> Taladrar un agujero de 1 cm en la parte inferior de una placa de cultivo de 35 mm. </li><li> Pegue el cubreobjetos recubiertos de poli-HEMA a la parte inferior de la placa de cultivo con Sylgard 184 (Dow Corning Co., Midland, MI) de manera que la cara cubierta de poli-HEMA se enfrenta hacia el interior del plato. Asegúrese de que el borde de la capa de poli-HEMA corre por el centro del agujero. Deje que la vajilla se seque durante 1-3 días a temperatura ambiente en un ambiente libre de polvo. </li><li> El principio de una línea en el exterior de el cubre con una pluma de punta de diamante siguiendo el borde de la capa de poli-HEMA. Entonces, el principio dos líneas que se cruzan en ángulos rectos a aproximadamente 2 mm de distancia. Los dos puntos de intersección servir de referencia o puntos fiduciales que se utilizarán para la reproducción de la misma orientación de la placa de cultivo en la platina del microscopio. </li><li> Esterilizar los platos bajo la luz ultravioleta durante la noche. </li><li> Para crear un medio de células adherentes de la placa de cultivo, la capa no poli-HEMA medio con Sal-1, que contiene un ratón anti-salamandra anticuerpo contra las membranas de células de la retina (generosamente proporcionada por el Dr. Peter MacLeish, Escuela de Medicina Morehouse , Atlanta, GA; ver MacLeish et al (1983)).. En primer lugar, cubrir el Sal-1 lados, con aproximadamente 100μl de 0,1 mg / ml estériles de cabra anti-ratón de anticuerpos IgG (Boehringer Mannheim Corporation, Indianapolis, IN), teniendo cuidado de no derramar el líquido sobre el lado en el poli-HEMA. Dejar durante tres horas, y luego retírela. Lave el área con la misma solución de Ringer estéril una vez o dos veces, luego coloque 100μl Sal-1 sobrenadante, teniendo cuidado de no derramar el líquido en la parte de poli-HEMA del plato. El primer anticuerpo asegura Sal-1 está revestido en una concentración conocida. </li><li> Incubar los platos con la noche a la mañana Sal-1. </li><li> Eliminar la solución de Sal-1 anticuerpo y lavar el área con solución de Ringer estéril. Introducir 2 ml de medio para el plato justo antes de la chapa de la célula. </li></ol> La composición de la solución de Ringer anfibio, medio libre de suero de anfibios, y la solución de Ringer para la digestión enzimática se encuentran en MacLeish y Townes Anderson (1988) y Mandell et al. (1993). Preparación de cultivos celulares Las células utilizadas en este video se deriva de la luz-adaptado, adultos, acuáticos fase de salamandras tigre (Ambystoma tigrinum), que mide 17-22 cm de longitud (Charles Sullivan Inc., en Nashville, TN), mantenido a 5 ° C en un 12 h: 12 h ciclo luz-oscuridad. Los protocolos fueron aprobados por el Comité Institucional y el empleo Comisión (IACUC) de la Universidad de Medicina y Odontología de Nueva Jersey, en estricta conformidad con las directrices de los Institutos Nacionales de Salud. Los procedimientos para la preparación de los cultivos de células retinianas son descritos por Nachman-Clewner y Townes-Anderson (1996) y son los siguientes: <ol><li> Decapitar y la médula de los animales. Eliminar los ojos y diseccionar las córneas y el iris. La retina se separe un poco en el ocular. Mediante la colocación de una pinza curva debajo de la retina, saque las retinas. No es importante si la lente se adjunta a la retina en esta etapa. </li><li> Recopilación de las retinas de 14 U / ml papaína (Worthington, Freehold, NJ) en solución de Ringer salamandra durante 40 minutos. </li><li> Antes de su uso, la solución de Ringer papaína debe ser burbujeado con 95% O 5.2% de gas CO 2 durante 30 min., Se esteriliza por el paso a través de un filtro de 0,22 micrones (Millipore, Carrigtwohill, Irlanda). </li><li> Enjuague las retinas con una solución estéril de Ringer salamandra tres veces. </li><li> Quitar las lentes de la solución de Ringer y suavemente triturar el tejido de la retina con un diámetro de 3 mm de pipeta para obtener una suspensión celular. </li></ol> Pinzas láser manipulación de neuronas aisladas <ol><li> Coloque el plato de la cultura en la platina del microscopio, la orientación de la antena mediante la alineación de una marca en el plato con un punto de referencia en el escenario. Guardar las coordenadas de los puntos de referencia en el ordenador. </li><li> Células de la placa en ambas Sal-1 y poli-HEMA mitad del plato. </li><li> Permitir que las células que conformarse con unos 20 minutos, que es lo suficientemente largo para que las células en el lado adherente del plato para insertarse en el sustrato de anticuerpos. </li><li> Seleccione una celda en el lado de Sal-1 adherente del plato. Marcar la X y la etapa y las coordenadas de una posición cercana a la célula. En nuestro caso, el punto fue de aproximadamente 10μm de las dendritas primarias de la celda seleccionada. Guardar las coordenadas en la computadora. </li><li> Seleccione una segunda neurona, en nuestro caso un fotorreceptor, en el lado del poli-HEMA de la antena y el uso de la herramienta pinzas láser para atrapar. Trampas se puede lograr en una amplia gama de niveles de potencia. Sin embargo, se establece la potencia del láser en un 10% -20%, que es lo suficientemente baja como para evitar los restos de captura junto con la célula, pero no suficiente para el transporte de la célula a través del medio. </li><li> Mientras mantiene la celda en la trampa, baje el escenario para que la célula está muy por encima de la superficie de la placa de cultivo y, sobre todo las neuronas adjunto. Mover el escenario bajo control de computadora para llevar el celular a la guardada previamente coordenadas X e Y en el Sal-1 lado. El movimiento en el escenario se fijó en 8-20μm / s, pero la velocidad máxima debe ser determinada empíricamente. A la llegada al punto de destino en el Sal-1 lado, aumentar la etapa para que la célula atrapada en la superficie del plato. La colocación de células se pueden hacer con una precisión de unas pocas micras. Permiten a la célula a adherirse a la Sal, un sustrato. </li><li> La obtención de imágenes digitalizadas de las dos células de la pareja antes y después de la formación par proporciona un registro útil. </li><li> Mantenga los pares de células en una incubadora. Para las células de salamandra, el lugar de los platos en una cámara húmeda a 10 º C. Las células pueden ser controlados por el transcurso de vídeo en tiempo y utilizar en cualquier técnica de las células biológicas requeridas. </li></ol>

<ol>
	<li>Ashkin, A., Dziedzic, J. M., Bjorkholm, J. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observation+of+a+Single-Beam+Gradient+Force+Optical+Trap+for+Dielectric+Particles.">Observation of a Single-Beam Gradient Force Optical Trap for Dielectric Particles.</a> Opt. Lett. 11, 288-290 (1986).</li><li>Clarke, R. J., Hoegnason, K., Brimacombe, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cone+and+rod+cells+have+different+target+preferences+in+vitro+as+revealed+by+optical+tweezers.">Cone and rod cells have different target preferences in vitro as revealed by optical tweezers.</a> Mol. Vision. 14, 706-720 (2008).</li><li>Folkman, J., Moscona, A. <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+cell+shape+in+growth+control.">Role of cell shape in growth control.</a> Nature. 273, 345-349 (1978).</li><li>Liu, Y., Cheng, D. K., Soneck, G. J., Berns, M. W., Chapman, C. F., Tromberg, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+localized+cell+heating+induced+by+infrared+optical+tweezers.">Evidence of localized cell heating induced by infrared optical tweezers.</a> Biophysical Journal. 68, 2137-2144 (1995).</li><li>MacLeish, P. R., Barnstable, C. J., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+monoclonal+antibody+as+a+substrate+for+mature+neurons+in+vitro.">Use of a monoclonal antibody as a substrate for mature neurons in vitro.</a> Proc. Natl. Acad. Sci. USA. 80, 7014-7018 (1983).</li><li>MacLeish, P. R., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+synapse+formation+among+major+classes+of+adult+salamander+retinal+neurons+in+vitro.">Growth and synapse formation among major classes of adult salamander retinal neurons in vitro.</a> Neuron. 1, 751-760 (1988).</li><li>Mandell, J. W., MacLeish, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Townes-Anderson+E.+Process+outgrowth+and+synaptic+varicosity+formation+by+adult+photoreceptors+in+vitro.">Townes-Anderson E. Process outgrowth and synaptic varicosity formation by adult photoreceptors in vitro.</a> J. Neurosci. 13, 3533-3548 (1993).</li><li>Nachman-Clewner, M., Townes-Anderson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury-induced+remodelling+and+regeneration+of+the+ribbon+presynaptic+terminal+in+vitro.">Injury-induced remodelling and regeneration of the ribbon presynaptic terminal in vitro.</a> J. Neurocytol. 25, 597-613 (1996).</li><li>Townes-Anderson, E., St Jules, R. S., Sherry, D. M., Lichtenberger, J., Hassanain, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromanipulation+of+retinal+neurons+by+optical+tweezers.">Micromanipulation of retinal neurons by optical tweezers.</a> Mol. Vis.. 4, (1998).</li></ol>

La luz tiene un momento, y cuando un rayo de luz se refracta al pasar a través de un celular, se requiere una fuerza para cambiar la dirección del momento. Debido a la ley de conservación del momento, una fuerza en la dirección opuesta que, a su vez, reacciona de nuevo en una celda. Ashkin (1991) demostró que la fuerza generada por un rayo láser enfocado por una lente objetivo del microscopio se mueve una celda hacia el centro de atención. A pesar de que un rayo láser genera sólo un piconewtons pocos de la fuerza, esta fuerza es su...

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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>25mm circle No.1 coverglass</td>
<td>&nbsp;</td>
<td>VWR Scientific Inc., Westchester, PA</td>
<td>48380 080</td>
<td>&nbsp;</td>
<tr>
<td>poly-2-hydroxyethylmethacrylate (poly-HEMA)</td>
<td>&nbsp;</td>
<td>Sigma Chemical Co., St Louis, MO</td>
<td>P-3932</td>
<td>Dissolve in 95% ethanol</td>
</tr>
<tr>
<td>Goat anti-mouse IgG antibody</td>
<td>&nbsp;</td>
<td>Chemicon International, Temecula CA</td>
<td>AP181</td>
<td> 1mg in 1ml, dilute 10x for use</td>
</tr>
<tr>
<td>Sal-1 supernatant containing mouse anti-salamander antibody</td>
<td>&nbsp;</td>
<td>generously provided by Dr. Peter MacLeish</td>
<td>&nbsp;</td>
<td>Dr. Peter MacLeish, Morehouse School of Medicine, Atlanta, GA</td>
</tr>
<tr>
<td>3 mm bore 5ml pyrex disposable pipets</td>
<td>&nbsp;</td>
<td>Corning Inc., Corning NY</td>
<td>7078A-5</td>
<td>&nbsp;</td>
<tr>
<td>Cell culture dishes 35mm x 10mm</td>
<td>&nbsp;</td>
<td>Corning inc., Corning NY</td>
<td>430165</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 silicone elastomer kit</td>
<td>&nbsp;</td>
<td>Dow Corning Corp., Midland MI</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Optical tweezers-microtool or laser tweezers</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 W continuous wave diode laser of 980nm wavelength</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc., Albuquerque NM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Axiovert 100 inverted light microscope</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>40x oil immersion plan neofluor objective lens</td>
<td>&nbsp;</td>
<td>Carl Zeiss Inc., Thornwood, NY</td>
<td>&nbsp;</td>
<td>Numerical aperture (N.A. 1.3)</td>
</tr>
<tr>
<td>Black and white CCD camera</td>
<td>&nbsp;</td>
<td>Sony Corporation, Tokyo, Japan</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Computer and joystick with software</td>
<td>&nbsp;</td>
<td>Cell Robotics Inc.</td>
<td>&nbsp;</td>
<td>for controlling a motorized stage</td>
</tr>		</tbody>
		</table>
		</div>

using laser tweezers for manipulating isolated neurons in vitro

En este documento y de vídeo, se describen los protocolos utilizados en nuestro laboratorio para estudiar las preferencias de orientación de los procesos de regeneración celular de neuronas de la retina adulta in vitro. Procedimientos para la preparación de los cultivos de células retinianas comienzan con la disección, la digestión y la trituración de la retina, y terminar con el revestimiento de células aisladas de la retina en platos elaborados especialmente para su uso con pinzas láser. Estos platos se dividen en un medio adhesivo celular y una media de células repelente. El lado adhesivo celular está recubierta con una capa de Sal-1 anticuerpos, que proporcionan un sustrato sobre el que las células crecen. Otros sustratos adhesivos podrían ser utilizados para otros tipos de células. El lado repelente de la célula está recubierta con una fina capa de poli-HEMA. Las células plateado en la parte de poli-HEMA del plato son atrapados con las pinzas láser, transportados y luego se coloca junto a una celda de la Sal-1 lado para crear un par. Formación de grupos de células de cualquier tamaño, debería ser posible con esta técnica. &quot;Pinzas láser controlado por micromanipulación&quot; significa que el investigador puede elegir la que las células se muevan, y la distancia deseada entre las células pueden ser estandarizados. Debido a que el rayo láser pasa a través de superficies transparentes de la placa de cultivo, selección y colocación de células se llevan a cabo en un ambiente cerrado y estéril. Las células pueden ser monitoreados por video time-lapse y utilizar en cualquier técnica de las células biológicas requeridas. Esta técnica puede ayudar a las investigaciones de las interacciones célula-célula.

Watch this Scientific Journal Video about Using Laser Tweezers For Manipulating Isolated Neurons In Vitro at JoVE.com

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Este video describe la manipulación de cultivos de neuronas con pinzas láser in vitro.

Utilizando pinzas láser para manipular neuronas aisladas in vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

using-laser-tweezers-for-manipulating-isolated-neurons-in-vitro

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.3K vistas.  University of Medicine and Dentistry of New Jersey - UMDNJ. Este video describe la manipulación de cultivos de neuronas con pinzas láser in vitro.

Video: Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

La medición de la exocitosis en las neuronas de un etiquetado FM

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Investigación

Biología

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

Los microchips de silicio para la Manipulación de la interacción célula-célula

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Using Micro-Electro-Mechanical Systems (MEMS) to Develop Diagnostic Tools

Using Micro-Electro-Mechanical Systems MEMS to Develop Diagnostic Tools

Utilizando Micro-Electro-Mechanical Systems (MEMS) para desarrollar herramientas de diagnóstico

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

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 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

El aislamiento de los ribosomas polipéptidos nacientes Bound<em&gt; In vitro</em&gt; Para la identificación de sitios de traslación pausa a lo largo del ARNm

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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 ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

La manipulación de la murino Glándula Lagrimal

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

La Técnica del riñón perfundido aislado del ratón

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análisis de suspensiones celulares aisladas de tejidos sólidos por Spectral Citometría de Flujo

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

Utilizando pinzas láser para manipular neuronas aisladas in vitro

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