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.

Transcript

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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Journal (Biology)

Journal (Biology)

Single-cell Microinjection for Cell Communication Analysis

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