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.

1. Preparation of Cells
<ol>
	<li>Maintain a culture of a thymic epithelial cell line (IT76M1) or cell to be tested in an incubator (37&#176;C/5% CO2).</li>
	<li>Wash the cells with PBS 1x (repeat this item 3x).</li>
	<li>Add Trypsin to the cells for 5 min.</li>
	<li>Add medium (twice of the volume of trypsin added in item 1.3) with 10% FBS (fetal bovine serum) to the cells with trypsin and centrifuge (800 x g for 5 min).</li>
	<li>Count the cells in a hemocytometer.</li>
	<li>Adjust the density of cells acc...

<ol>
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In order to verify the presence of functional intercellular gap junction, the use of tracers, which are membrane impermeable, although permeable by intercellular channels are required16. Fluorescein, the first fluorescent dye to observe cell-to-cell coupling22, is permeable between non junctional membranes3 and has therefore been substituted by Lucifer Yellow dye15. Currently, to find the best choice among the many different t...

Gap junctions are intercellular channels that allow the intercommunication among neighboring cells1. This communication connects two or more neighboring cells, where each one contributes with a connexon or hemichannel to form the intercellular channel. In mammalian cells, the connexon is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm2. Gap junctions not only permit the flow of ions, second messengers and small metabolites, but also contribute to many forms of cellular communication in many physiological processes, such as synaptic transmission, heart contraction, cell growth and differentiation3,4,5,6,7,8. In addition gap junctions have been associated with many diseases including cancer9,10, muscular atrophy11, some genetic diseases and demyelinating diseases12.
This type of intercellular crosstalk can be evaluated by several methods13,14,15,16. In this paper, we show how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. We discuss how to prepare the cells and the micropipette, the usage of the micromanipulator and the injection of Lucifer Yellow dye in a thymic epithelial cell line. Usually, this experimental procedure could be analyzed by the average of connected cells to the cell loaded with dye. In addition, this method could be used with other fluorescent dyes with molecular weight below the gap junctions cut-off which is approximately 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.

Thymic epithelial cell line IT-76MI were used to evaluate dye coupling by gap junctions as these cells were described to express functional gap junctions formed by connexin 4321. Figure 1 shows the injection of Lucifer Yellow when applied in the one cell below the tip of the pipette. After few minutes, connected cells become fluorescent (asterisks) indicating the diffusion of the fluorescent dye through the gap junctions. The number of cells and time to became fluo...

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

Single-cell Microinjection for Cell Communication Analysis

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.

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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