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. Preparazione di cellule <ol><li> Mantenere una cultura di una linea timica cellule epiteliali (IT76M1) o cellule da testare in un incubatore (37 ° C / 5% di CO 2). </li><li> Lavare le cellule con PBS 1x (ripetere questo punto 3x). </li><li> Aggiungere tripsina alle cellule per 5 min. </li><li> Aggiungere mezzo (il doppio del volume di tripsina aggiunto al punto 1.3) con 10% FBS (siero bovino fetale) alle cellule con tripsina e centrifugare (800 xg per 5 min). </li><li> Contare le cellule in un emo...

<ol>
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Al fine di verificare la presenza di funzionali gap junction intercellulare, l&#39;uso di rivelatori, che sono membrana impermeabile, anche se permeabile da canali intercellulari sono necessari 16. Fluoresceina, il primo colorante fluorescente per osservare cellula-cellula di accoppiamento 22, è permeabile tra membrane non giunzionali 3 ed è stato quindi sostituito da Lucifer colorante giallo 15. Attualmente, per trovare la...

Giunzioni intercellulari sono canali che permettono l&#39;intercomunicazione tra cellule vicine 1. Questa comunicazione connette due o più celle confinanti, dove ognuno contribuisce con connessone o hemichannel per formare il canale intercellulare. In cellule di mammifero, la connessone è formata da sei connessine, monomeri con quattro domini transmembrana e un C e terminale N all&#39;interno del citoplasma 2. Giunzioni non solo permettono il flusso di ioni, secondi messaggeri e piccoli metaboliti, ma contribuiscono anche a molte forme di comunicazione cellulare in molti processi fisiologici, come la trasmissione sinaptica, contrazione cardiaca, la crescita e la differenziazione cellulare 3, 4, 5, 6, 7, 8. Inoltre giunzioni sono stati associati conmolte malattie tra cui il cancro 9, 10, 11 atrofia muscolare, alcune malattie genetiche e malattie demielinizzanti 12. Questo tipo di crosstalk intercellulare può essere valutata mediante diversi metodi 13, 14, 15, 16. In questo lavoro, si mostra come eseguire una microiniezione cella singola di Lucifero giallo di visualizzare la comunicazione cellulare attraverso gap giunzioni nelle cellule viventi. Discutiamo come preparare le cellule e la micropipetta, l&#39;uso del micromanipolatore e l&#39;iniezione di Lucifero colorante giallo in una linea di cellule epiteliali del timo. Solitamente, questa procedura sperimentale potrebbe essere analizzato dalla media delle celle collegate alla cella caricato con colorante. Inoltre, questo metodo può essere usato con altri coloranti fluorescenti con peso molecolare inferiore del gapgiunzioni cut-off, che è di circa 1.000 dalton.

<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.

Timica linea di cellule epiteliali IT-76MI sono stati utilizzati per valutare l&#39;accoppiamento colorante da giunzioni in quanto queste cellule sono state descritte per esprimere giunzioni funzionali formati da connessina 43 21. La Figura 1 mostra l&#39;iniezione di Lucifer Yellow quando applicato in una cella sotto la punta della pipetta. Dopo alcuni minuti, celle collegate diventano fluorescenti (asterischi) indica la diffusione del colorante fluorescente attr...

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

Single-cell Microinjection for Cell Communication Analysis

Descriviamo qui come eseguire una microiniezione cella singola di Lucifero giallo di visualizzare la comunicazione cellulare attraverso gap giunzioni nelle cellule viventi, e fornire alcuni suggerimenti utili. Ci aspettiamo che questo articolo vi aiuterà a tutti di valutare il grado di accoppiamento cellulare a causa di giunzioni funzionali. Tutto qui descritto potrebbe essere, in linea di principio, atto ad altri coloranti fluorescenti con peso molecolare inferiore a 1000 Daltons.

Microiniezione cella singola per l&#39;analisi cellulare comunicazione

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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