Name: single-cell microinjection for cell communication analysis
Uploaded: 2017-02-26T13:00:00
Description: Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

The authors dedicate this paper in honor of Prof. Gilberto Oliveira-Castro who introduced research in intercellular communication by gap junctions in Brazil. This work was funded by Capes, CNPQ and Faperj.

<html> 1. Preparación de las células <ol><li> Mantener un cultivo de una línea celular epitelial del timo (IT76M1) o células a ensayar en una incubadora (37 ° C / 5% de CO 2). </li><li> Se lavan las células con PBS 1x (repetir este artículo 3x). </li><li> Añadir tripsina a las células durante 5 min. </li><li> Añadir medio (dos veces del volumen de tripsina añadida en el punto 1.3) con 10% de FBS (suero bovino fetal) a las células con tripsina y se centrifuga (800 x g durante 5 min). </li><li> Con...

<ol>
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Con el fin de verificar la presencia de brecha de la salida intercelular funcional, el uso de trazadores, que son de la membrana impermeable, aunque permeable por canales intercelulares se requieren 16. Fluoresceína, el primer tinte fluorescente para observar célula a célula de acoplamiento 22, es permeable entre las membranas no de unión 3 y por lo tanto ha sido sustituido por Lucifer colorante amarillo 15. En la actuali...

Gap cruces son canales intercelulares que permiten la intercomunicación entre las células vecinas 1. Esta comunicación se conecta dos o más células vecinas, donde cada uno contribuye con una conexón o hemicanal para formar el canal intercelular. En células de mamíferos, la conexón está formado por seis conexinas, monómeros con cuatro dominios transmembrana y una C y N terminal dentro del citoplasma 2. Gap cruces no sólo permiten el flujo de iones, segundos mensajeros y pequeños metabolitos, sino que también contribuyen a muchas formas de comunicación celular en muchos procesos fisiológicos, tales como la transmisión sináptica, la contracción del corazón, el crecimiento y la diferenciación celular 3, 4, 5, 6, 7, 8. Además las uniones comunicantes se han asociado conmuchas enfermedades incluyendo el cáncer de 9, 10, atrofia muscular 11, algunas enfermedades genéticas y enfermedades desmielinizantes 12. Este tipo de diafonía intercelular se puede evaluar mediante varios métodos 13, 14, 15, 16. En este artículo se muestra cómo realizar una microinyección de una sola célula de Lucifer amarillo para visualizar la comunicación celular a través de las uniones gap-en las células vivas. Se discute cómo preparar las células y la micropipeta, el uso de la micromanipulador y la inyección de un colorante amarillo Lucifer en una línea de células epiteliales del timo. Por lo general, este procedimiento experimental podría ser analizado por el promedio de células conectadas a la célula de carga con colorante. Además, este método podría ser utilizado con otros colorantes fluorescentes con peso molecular por debajo de la brechauniones de corte que es de aproximadamente 1.000 daltons.

<table border="0" cellpadding="0" cellspacing="0" width="1228">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Lucifer yellow</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">L0259</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Lithium Chloride</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">L4408</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS tablets</td>
			<td style="width:71px;">Sigma&#160;</td>
			<td style="width:137px;">P4417</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">RPMI</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">R4130</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Bovine fetal serum</td>
			<td style="width:71px;">Cultilab</td>
			<td style="width:137px;"></td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Trypsin</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">T4799</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:137px;">TE-2000</td>
			<td>For microinjection experiments, one needs an inverted fluorescence microscope and &#64257;lters for &#64258;uorescent microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vibration-insulated table&#160;</td>
			<td style="width:71px;">Newport</td>
			<td style="width:137px;">VH3036W-OPT</td>
			<td>A vibration-insulated table is needed to protect the experiments from vibration and avoid cell damage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:137px;">MMO-203</td>
			<td>This equipment allows precision adjustments of the micropipette, which is needed for cell micro injection.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">Current Generator&#160;</td>
			<td style="width:71px;">Digitimer</td>
			<td style="width:137px;">DS2</td>
			<td>To produce the dye flow through the micropipette, a current below one nano ampere was given using a current generator with an electrode inside the micropipette or an amplifier which has a capacitance compensation circuit (old electrometer) or current injection functions of new patch clamp amplifiers, and the ground wire submersed in the plate dish. Alternatively, the dye can be injected by a pneumatic microinjector, following the factory recommendations.&#160;&#160;&#160;</td>
		</tr>
	</tbody>
</table>

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.

Línea de células epiteliales del timo IT-76MI se utilizaron para evaluar tinte de acoplamiento por uniones comunicantes como se describen estas células para expresar uniones gap funcionales formados por conexinas 43 21. La Figura 1 muestra la inyección de Lucifer Yellow cuando se aplica en la célula debajo de la punta de la pipeta. Después de algunos minutos, las células se vuelven conectados fluorescente (asteriscos) que indica la difusión del colorante f...

Watch this Scientific Journal Video about Single-cell Microinjection for Cell Communication Analysis at JoVE.com

Single-cell Microinjection for Cell Communication Analysis

Se describe aquí cómo realizar una microinyección de una sola célula de Lucifer amarillo para visualizar la comunicación celular a través de las uniones gap-en las células vivas, y proporcionar algunos consejos útiles. Esperamos que este documento ayudará a cada uno para evaluar el grado de acoplamiento celular debido a uniones gap funcionales. Todo lo descrito aquí podría ser, en principio, adaptado a otros colorantes fluorescentes con peso molecular inferior a 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 ...

Hi, my name is Anael. I work at Oswaldo Cruz Foundation at Cellular Communication Laboratory. We study here P2 Receptors and gap injection in the context of immune system and liver.Today, I will present you the microinjection of cell. Gap injections play some physiological roles as synapse transmission, heart contractions, and others. For this technique, we can study if the gap injections are functional or not.Okay, let's see the base components of the microinjection setup. Here we have the inverted fluorescence microscope where we will see the cells. This is a CCD camera to record the results.We have here the current generator that we will introduce the dye into the cell. Next we have the holder where the microelectrode is connected. And the micro-manipulator that's connected to the holder and allow the movement of the microelectrode in the three axis, Z, Y, and X.Before start the experiment, we have to make the microelectrode.We use this borosilicate glass capillary and this equipment named polar. We attach the borosilicate capillary here. This tungsten filament we will heat to make two microelectrodes.Now the tungsten filament's heating and the wave here we will pull down to make the two microelectrodes.Easy. Our first step is to fill the microelectrode with the dye. One tip here is that you have to fill only the tip with a few microliters to do the experiment.It's not necessary to fill everything. Note with more details. Fill only the tip.Let's start an experiment. We have here the cells in the petri dish, in a sodium solution. The silver wire connected to the current generator.We have now to attach the microelectrodes in the head stage. Here in the head stage we have another silver wire also connected to the current generator. Once the microelectrode is attached, we can put it inside of the bath.Note that the tip of the pipette is carefully touching the bath. This procedure is make very carefully to avoid the crash of the tip in the bottom of the petri dish. Once inside of the bath, we can go to the microscope to look for the shadow of the micropipette.We recommend we start looking in the magnification lens. When we find the shadow of the pipette, we can move the microelectrode toward the cell using the micromanipulator. Once it's very close to the cell, we can do a test post to verify if the microelectrode's not obstructed.How we will see next. Here we are seeing the micropipette and we are adjusting the micropipette very close to the cell. We can see cell movement right and left.Then we change to the filter, fluorescence filter. We can apply hyperpolarizing pulse to test if the tip of the pipette is not obstructed. Since the micropipette is inside of the cell, we can apply hyperpolarizing pulse to load the cell with the dye.We use here the loose free yellow dye. After loading the cell with the dye, if the neighbor cells are connected by gap injections, we wait some minutes and we will see neighbor cells brightening by the diffusion of the dye by these gap injections. In our experiment we can see five cells, marked with stars, showing fluorescence.Here there is a good example of an experiment of microinjection in thymic epithelial cells, in A and B.In the figures C and D, shows a microinjection in thymic inert cell of loose free yellow and another dye, not permeable to gap injection showed in the insert. The figure E and F thymic epithelial cell line microinjected with loose free yellow and in the insert loose free yellow and a gap injection block. The results show that loose free yellow did not spread to the neighbor cells.Next we see a microinjection in thymic epithelial cells, in presence of dexamethasone and the quantification in B.Dexamethasone increased the degree of coupling, as showed in the figure B.Of 100 injections, the number of three or four cells connected was more frequent in the presence of this drug. I hope I could help beginners to do this important technique to cellular communication study. Thanks and bye bye.

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