Los autores reconocen programa de investigación creativa de la Universidad de Clemson y NSF CBET1254609 para proporcionar fondos para este proyecto.

1. La producción de una Cámara de microcanales para crear un gradiente de concentración <ol><li> Cortar una pieza de molde de acrílico <ol><li> Obtener una pieza de acrílico de la anchura deseada. Típicamente utilizar hojas de acrílico 1/16 pulgadas de espesor. hojas más gruesas son difíciles de cortar bien y muy delgadas hojas no tienen la resistencia mecánica, provocando su rotura o deformación durante el proceso. </li><li> Crear un archivo CAD con la forma deseada de la pieza de acrílico para producir una cavidad dentro del polidimetilsiloxano (PDMS). Ajustar la anchura del cauce y longitud para alcanzar el gradiente deseado correspondiente a los protocolos experimentales individuales. <ol><li> A continuación, utilice una pieza de acrílico de las siguientes dimensiones: dos cuadrados (0,254 cm x 0,254 cm) conectados por un canal de ancho de 0,127 cm (0,254 cm de largo) (Figura 1). </li></ol></li><li> Importe el archivo CAD en el aparato de corte por láser y colocar la pieza de acrílico en un cortador de láser de acuerdo con la manuel protocolo del cante. NOTA: Como alternativa, 3D-imprimir la pieza desde el diseño CAD. La ventaja de este método es que los materiales alternativos pueden utilizarse para el molde; Sin embargo, la resolución y la reproducibilidad se pueden disminuir con la impresión en 3D en comparación con el corte por láser. </li></ol></li></ol><img alt="Figura 1" src="/files/ftp_upload/55264/55264fig1.jpg" /> Figura 1: (CAD) Representación Computer-Aided Design de la Cámara de microcanales. Esta imagen representa el dibujo CAD de la pieza de acrílico necesaria para crear la cámara de microcanales. 1 A) Vista superior de dibujo CAD, 1B) Vista lateral de CAD dibujo con dimensiones en pulgadas, 1C) Vista superior de CAD dibujo con dimensiones en pulgadas <a href="http://ecsource.jove.com/files/ftp_upload/55264/55264fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <ol start="2"><li&gt; Verter el polidimetilsiloxano (PDMS) <ol><li> En un recipiente para pesar, mezclar 10 partes en peso de base de elastómero comercial (por ejemplo Sylgard 184) con 1 parte en peso de elastómero comercial Agente de curado (por ejemplo Sylgard 194). Típicamente, la mezcla 10 g de elastómero base a 1 g de elastómero agente de curado. </li><li> Agitar para combinar la base y agente de curado con una micropipeta para 5 min. </li><li> Configurar una campana de vacío y colocar el barco sopesar con PDMS en el vacío durante 30 min para eliminar las burbujas de aire atrapadas. </li><li> Apague el vacío y retire el recipiente para pesar. Verter el PDMS en la parte superior del acrílico recortada en una pequeña placa de Petri. Asegúrese de que el PDMS cubre completamente el inserto. </li><li> Permitir a los PDMS curar durante la noche (al menos 18 h) a temperatura ambiente. </li></ol></li><li> Extracción de la inserción de PDMS <ol><li> Después de curar el PDMS, extraiga la tapa cortando cuidadosamente el PDMS alrededor de la pieza de acrílico con un bisturí. </li></ol&gt; </li></ol> 2. placas las células en una Cámara de microcanales <ol><li> La esterilización de la cámara para el recubrimiento de las células. NOTA: Mientras PDMS pueden ser esterilizados con óxido de etileno u otras técnicas, un tratamiento UV rápida es rápido, barato, y suficiente para estudios de cultivo celular. La duración del tratamiento UV corta no afectó la estructura o la integridad mecánica de la pieza de PDMS. <ol><li> En una cabina de cultivo de células estándar, cubrir completamente la cámara con 70% de etanol. </li><li> Ponga la placa de Petri en la campana de flujo laminar con las tapas. </li><li> Cierre el capó y encender la luz UV. Exponerlo a la luz UV durante 1-2 h. </li><li> Abra la campana de flujo laminar y esperar 15 minutos para restablecer el flujo. </li><li> Retire el etanol al 70%. </li><li> Lavar las cámaras dos veces con solución salina estéril tamponada con fosfato (PBS). Use 1 ml de PBS para cada lavado. Retirar PBS y se permiten cámaras que se seque durante la noche con las tapas. NOTA: Como alternativa, en lugarde utilizar una campana de flujo laminar, usar una caja de cámara UV para la esterilización si hay alguno disponible. Hacer una caja de UV mediante la colocación de una fuente de luz UV en la parte posterior de una caja de cartón forrada con papel de aluminio. El papel de aluminio refleja la luz para permitir la iluminación uniforme de la muestra. </li></ol></li><li> Sembrando células en la Cámara <ol><li> Contar las células usando azul tripán y un hemocitómetro. <ol><li> Añadir 100 ml de la suspensión celular a 400 l de 0,4% Trypan azul y mezclar suavemente. Aplicar 100 l de esta solución a la hemocitómetro rellenando suavemente ambas cámaras bajo el cubreobjetos de vidrio. </li><li> Utilizar un microscopio con un objetivo de 10X para centrarse en la cuadrícula en el hemocitómetro. Utilizar un contador de cuenta de la mano para contar las células no teñidas en vivo dentro de un conjunto de 16 plazas en el hemocitómetro. </li><li> Recuento de células en los 4 grupos de 16 cuadrados. Tome el recuento medio de células a partir de las 4 series de 16 plazas y se multiplica por 4 5x10. Este número final es el number de células / ml en la suspensión celular. </li></ol></li><li> Añadir 2.500 células a un pocillo de cada cámara. Esto se traduce en una densidad de células casi confluentes de ~ 30.000 / cm 2. </li><li> Permiten a las células para sentarse en la cámara durante 60 minutos antes de la adición de medios (Modificado de Dulbecco del medio Eagle, 10% de suero fetal bovino, 1% de penicilina-estreptomicina). </li></ol></li></ol> 3. Los bloques de agarosa dextrano empapados <ol><li> Creación de un bloque de agarosa <ol><li> Verter 3% de agarosa en el molde impreso 3D (2 mm x 2 mm x 2 mm). </li><li> Dejar que la agarosa solidifique en una cámara de vacío durante 30 min para eliminar las burbujas. </li><li> Retire el bloque de agarosa del molde. NOTA: Como alternativa, se vierte 3% de agarosa en pequeña placa de Petri en un espesor de 2 mm. Corte un bloque de 2 mm x 2 mm x 2 mm con un bisturí u otra herramienta de corte. Esto no es tan preciso como el sistema de molde, pero puede ser un poco más fácil de hacer. </li></ol></li><li> completamente submerge el bloque de agarosa en una solución de la concentración deseada del factor quimiotáctico. Para evaluar la formación gradiente de concentración, lave la cámara en primer lugar con dextrano fluorescente. La concentración y peso molecular del dextrano deben coincidir con la de un factor o fármaco de interés soluble. </li><li> Remojar la noche a la mañana de bloques de agarosa. </li></ol> 4. lapso de tiempo de la difusión de dextrano para evaluar el gradiente de concentración del factor soluble <ol><li> Coloque el bloque de agarosa dextrano empapado en un pequeño pozo cuadrado de la cámara de microcanales. </li><li> Coloque la cámara de microcanal en un sistema de imágenes de células o utilizar otro microscopio de fluorescencia con una función de tiempo. Comienza el lapso de tiempo de acuerdo a las instrucciones del fabricante. Nota: Los resultados mostrados son tomadas con filtro de GFP, el 50% de luz, 4/10 pH, y la magnificación 4X. </li></ol> 5. lapso de tiempo de Crecimiento Celular <ol><li> células de la placa en la cámara de microcanal en un extremo de lacámara. Aquí, utilizar fibroblastos 3T3. Añadir 2.500 células a un pocillo en la cámara de microcanal. Recuento de células como se describe en 2.2.1. <ol><li> Permiten a las células para sentarse en la cámara durante 60 minutos antes de la adición de medios (Modificado de Dulbecco del medio Eagle, 10% de suero fetal bovino, 1% de penicilina-estreptomicina). Mantener la cámara de microcanal con células cultivadas en placas a 37 ° C en 5% de CO 2. La migración celular a través del canal se pueden ver con un microscopio. </li><li> Añadir un marcador o utilizar un defecto microscópico en la cámara como un marcador con el fin de servir como un punto de partida para cuantificar la distancia los delanteros célula se mueve. </li></ol></li><li> Establecer un gradiente mediante la colocación de un gel de agarosa (8 mm 3 bloques) que contiene el factor de crecimiento concentrado, quimio-atrayente, drogas, u otro factor está probando (aquí, 40% de FBS se utiliza como un ejemplo) en un extremo del microcanal cámara. </li><li> Tomar imágenes con lapso de tiempo de la parte frontal de células en movimiento. Aquí, los resultados muestran imágenes de la célula front que fueron tomadas cada 12 h utilizando un sistema de imagen. </li><li> Use imágenes de la parte delantera celular para cuantificar la tasa de crecimiento celular. Calcular la tasa de crecimiento por primera utilizando la función polyfit MATLAB (roipoly) para crear una máscara poligonal definida por el frente de células, las paredes del canal, y el marcador de referencia. Las dimensiones de esta máscara producen la distancia de migración de la parte frontal de la que se calcula la velocidad del frente de celda promedio. Otros estudios han utilizado un método similar 8. NOTA: Comparar el borde de la parte delantera de crecimiento de células en cada trama a la concentración del factor soluble en esa misma distancia. El gradiente de concentración factor soluble se calcula usando un modelo de difusión 1D aproximación. </li><li> Tomar imágenes fluorescentes de las células por medio de faloidina y DAPI. <ol><li> Fijar las células antes de la tinción con faloidina. <ol><li> Cálido paraformaldehído al 4% a 37 ° C. </li><li> Agregar suficiente paraformaldehído al 4% para cubrir las células. Mantener células en elde paraformaldehído durante 10 min. </li><li> Enjuague dos veces con PBS durante 15 min cada vez. Use suficiente PBS para cubrir las células. </li></ol></li><li> Retirar PBS a partir de células y añadir solución permeabilizante suficiente (0,1% Triton X-100 en PBS) para cubrir células. Deje la solución durante 10 min. </li><li> Lavar dos veces con PBS durante 5 min cada vez. Use suficiente PBS para cubrir las células. </li><li> Retirar PBS a partir de células y añadir solución de (1% de albúmina de suero bovino en PBS) de bloqueo para 20 min. </li><li> Eliminar la solución de bloqueo y se lava 2 veces con PBS durante 5 minutos cada vez. Use suficiente PBS para cubrir las células. </li><li> A partir de este punto, proteger a las células de la luz con papel de aluminio cuando no se utilicen. Retirar PBS a partir de células y añadir faloidina 488 diluido 1:50 en PBS durante 1 h. </li><li> Lavar 2 veces en PBS durante 5 min para cada lavado. Use suficiente PBS para cubrir las células. A continuación, retire PBS a partir de células. </li><li> Añadir DAPI (4 &#39;, 6-diamidino-2-fenilindol) diluido 1: 1000 en PBS a las células durante 15 min. </li><li> Retire y DAPILavar las células dos veces con PBS durante 5 min para cada lavado. </li><li> Retire último lavado y añadir una cantidad suficiente para cubrir las células PBS. Las células ahora se pueden obtener imágenes con un microscopio de fluorescencia. </li></ol></li></ol>

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(69), (2012).</li></ol>

Los autores no tienen nada que revelar.

Nuestra cámara de microcanal puede ser utilizado para una multitud de propósitos, incluyendo la determinación de la tasa de migración celular en respuesta a factores de crecimiento y factores quimiotácticos y la medición de la velocidad de difusión de un fármaco a partir de una matriz de polímero. Es posible utilizar nuestra cámara de microcanal para hacer crecer células y colocar un quimioatrayente en un extremo de la cámara. Las células crecen en respuesta a la quimioatrayente, y la migración de las cél...

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Sigma-Aldrich</td>
			<td>761036</td>
			<td>poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent</td>
		</tr>
		<tr>
			<td>Acrylic Sheets</td>
			<td>US Plastic</td>
			<td>44200</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes&#160;</td>
			<td>Falcon&#160;</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD20s-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose, Type I, Low EEO</td>
			<td>Sigma-Aldrich</td>
			<td>A6013-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>11965092</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serium</td>
			<td>Fisher Scientific</td>
			<td>16000036</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15140148</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP24384</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td>AME3300</td>
			<td>Instrument used for taking time-lapse images</td>
		</tr>
		<tr>
			<td>Versa LASER</td>
			<td>Universal Laser Systems, Inc.&#160;</td>
			<td>Model number VLS2.30</td>
			<td>Laser cutter used for cutting plastic</td>
		</tr>
	</tbody>
</table>

a customizable chamber for measuring cell migration

La migración celular es una parte vital de la respuesta inmune, el crecimiento y la cicatrización de heridas. La migración celular es un proceso complejo que implica interacciones entre las células, la matriz extracelular, y factores químicos solubles y no solubles (por ejemplo, factores quimiotácticos). Los métodos estándar para la medición de la migración de las células, tales como el ensayo de cámara de Boyden, el trabajo contando las células a ambos lados de un divisor. Estas técnicas son fáciles de usar; Sin embargo, ofrecen poca modificación geométrica para diferentes aplicaciones. En contraste, los dispositivos de microfluidos pueden ser utilizados para observar la migración de células con gradientes de concentración personalizables de factores solubles 1, 2. Sin embargo, los métodos para hacer los ensayos de microfluidos basados ​​pueden ser difíciles de aprender. A continuación, describimos un método fácil para la creación de cámaras de cultivo de células para medir la migración de células en respuesta a gradientes de concentración químicos. Nuestros millas celularesmétodo de la cámara gración puede crear diferentes gradientes de concentración lineal con el fin de estudiar la migración celular para una variedad de aplicaciones. Este método es relativamente fácil de usar y se lleva a cabo normalmente por los estudiantes universitarios. La cámara de microcanal fue creado por la colocación de un inserto acrílico en la forma de la cámara de microcanal final por pocillo en una placa de Petri. Después de esto, poli (dimetilsiloxano) (PDMS) se vertió en la parte superior del inserto. El PDMS se deja endurecer y después se eliminó la inserción. Esto permitió la creación de los pozos en cualquier forma o tamaño deseado. Las células se pueden añadir posteriormente a la cámara de microcanal, y agentes solubles se pueden añadir a uno de los pozos por remojo un bloque de agarosa en el agente deseado. El bloque de agarosa se añade a uno de los pozos, y las imágenes de lapso de tiempo puede ser tomada de la cámara de microcanal con el fin de cuantificar la migración celular. Las variaciones de este método se pueden hacer para una aplicación determinada, por lo que este método higHly personalizable.

La figura 2 muestra el movimiento de la parte delantera de células a través del canal en respuesta a un gradiente de suero bovino fetal colocado en el extremo opuesto del canal, desde donde se siembran las células. El frente celular se muestra a las 48 h (figura 2A), 72 h (Figura 2B), 96 h (Figura 2C), y 120 h (Figura 2D) post chapado. El movimiento de la parte frontal de células fue rastreado con es...

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A Customizable Chamber for Measuring Cell Migration

Este protocolo detalla un método adaptable para medir la migración de células en respuesta a quimioatrayentes, que también pueden ser utilizados para determinar la velocidad de difusión de un fármaco fuera de una matriz de polímero.

Una Cámara personalizable para medir la migración celular

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

a-customizable-chamber-for-measuring-cell-migration

Research

JoVE Journal

Biology

10.5K vistas.  Clemson University.  Este protocolo detalla un método adaptable para medir la migración de células en respuesta a quimioatrayentes, que también pueden ser utilizados para determinar la velocidad de difusión de un fármaco fuera de una matriz de polímero. 

Video: A Customizable Chamber for Measuring Cell Migration

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

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process 1. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port 2. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.

We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.

Estimulación Cerebral sector mediante una red de microfluidos y de la Cámara de perfusión estándar

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Imágenes en directo de la migración de células gliales en el ojo del disco imaginal de Drosophila

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

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 ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

Un ensayo para medir la actividad de Escherichia coli Inducible lisina Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Microscopia intravital de la microcirculación en el músculo de ratón Cremaster para el análisis de la migración de la célula madre periféricas

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

La microinyección de una sola célula de Análisis de Comunicación Móvil

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of 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 ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

Filamentos intermedios y microtúbulos con microscopía de reconstrucción óptica estocástica directo 2-dimensional de imágenes

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Cell migration is an important process that influences many aspects of health, such as wound healing and cancer, and it is, therefore, crucial for developing methods to study the migration. The scratch assay has long been the most common in vitro method to test compounds with anti- and pro-migration properties because of its low cost and simple procedure. However, an often-reported problem of the assay is the accumulation of cells across the edge of the scratch. Furthermore, to obtain data from the assay, images of different exposures must be taken over a period of time at the exact same spot to compare the movements of the migration. Different analysis programs can be used to describe the scratch closure, but they are labor intensive, inaccurate, and forces cycles of temperature changes. In this study, we demonstrate an optimized method for testing the migration effect, e.g. with the naturally occurring proteins Human- and Bovine-Lactoferrin and their N-terminal peptide Lactoferricin on the epithelial cell line HaCaT. A crucial optimization is to wash and scratch in PBS, which eliminates the aforementioned accumulation of cells along the edge. This could be explained by the removal of cations, which have been shown to have an effect on keratinocyte cell-cell connection. To ensure true detection of migration, pre-treating with mitomycin C, a DNA synthesis inhibitor, was added to the protocol. Finally, we demonstrate the automated optical camera, which eliminates excessive temperature cycles, manual labor with scratch closure analysis, while improving on reproducibility and ensuring analysis of identical sections of the scratch over time.

Optimized Scratch Assay for In Vitro Testing of Cell Migration with an Automated Optical Camera

Here we describe a procedure to test the cell migration in vitro with an automated optical camera microscope. The scratch assay has been widely used where the closure of the scratch is followed for a set period. The optical microscope enables dependable and cheap detection of cell migration.