A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

Electroporation is a technique where an electrical current is applied to live cells in order to open transient pores on the cell membrane. Through these pores, dyes and other molecules that normally do not cross the cell membrane can enter the cell the pores reclose quickly, with no damage to the cell. We will describe how a adhere cells can be electroporated as they're growing on a slide that is coated in part with electrically conductive and transparent material.

The material is indium tin oxide, and as it conducts electrical current, it opens pores that allow a dye to enter the cell. Then it is possible to examine if the cells have gap junctions or not, and how many gap junctions are small channels that connect the interiors of neighboring cells and allow the passage of small molecules. It is long been recognized that gap junctional communication has a variety of important functions in the cell, including neoplastic transformation and metastasis.

Welcome to this cell projects presentation about how to perform adherent cell electroporation using the Insitu. This image shows, which we'll be able to do, the fluorescent dye loose for yellow, was introduced into cells growing on a transparent and conductive glass. Up to this line, the line is easier to see under phase contrast.

Here it is. You can see that the dye has diffused through gap junctions from the cells on the left, which have been loaded with dye by electroporation to the cells on the right. Resulting in this Gradient of fluorescence cells are tryps.

Inized then transferred into a Tube in centrifuge. It is very important for the cells to be well spread as they grow in the chamber. For this reason, it is best to centrifuge the cells to remove any traces of trypsin.

The medium with traces of trypsin is suctioned off cells are resuspended in approximately one milliliter of Growth medium, and then plated in the chamber. The pipette contains the cell suspension, remove the lid from the Chamber and pipette the cells dropwise into the chamber to make it easier to carry around. The chamber is placed in a six centimeter Petri dish.

The chamber goes back in the incubator for the cells to grow. The plated cells will spread well and they will grow to touch each other and to form gap junctions. When the cells are ready to electro parade, set the insitu pulse generator to the desired conditions.

Refer to the manual for description of the simple setup process. When the voltage on the insitu is set, the red light on the keypad will turn on. Take the two warmed bottles of DMEM without calcium out of the water bath one is DMEM without calcium, but with dialyzed serum, and one is DMEM.

Without calcium, have the pipettes ready load looser for yellow into the pipetter. Instead of the sigh, take the cells out of the incubator. Remove the medium by inverting it in the wash beaker at first.

Then dabbing it onto the tissue wash once with DMEM without calcium, do not try to remove absolutely all of the DMEM because the cells may dry. Add the Lucifer yellow carefully onto the Barrier so as not to disturb the cells but quickly enough so that the cells do not dry. Use 200 microliters for each half of the chamber.

Put the lid on the insitu chamber, then Load it into the slider. Make sure that the end tabs of the chamber fit into the slots of the slider by pushing the slider. Insert the chamber into the chamber holder.

Push the pulse button on the pulse generator keypad. The LED on the pulse generator and on the chamber holder will flash red during the electroporation process and then flash green to indicate the init cycle is complete. Pull The slider back under the chamber holder and remove the chamber.

Aspirate most of the Lucifer yellow to use it again, but take care not to dry the cells. Add DMEM without calcium. With dialyzed serum, the serum helps the porous to close the chamber goes back into the incubator for the transfer Of Lucifer yellow through gap junctions to happen.

Timing must be consistent from Experiment to experiment. The standard is three minutes. Pour off the medium into the waste beaker.

Add 4%formaldehyde fixing solution with a paste or pipette, Wait at room temperature for at least two minutes. Pour off the formaldehyde and rinse a couple of times. With PBS, the cells are fixed to the glass, so there's little danger of them detaching during washing.

To be able to see the cells under phase contrast, it is important to place a cover slip on top of the chamber. Fill the chamber with PBS forming a proud meniscus. Avoiding air bubbles, place the cover slip.

Don't worry if the PBS overflows Just absorb the extra PBS onto tissue. You need an inverted fluorescent microscope, preferably with phase contrast and with a filter block for Lucifer yellow. Under phased contrast, look for the edge of the electroporated area.

You will see two lines Under fluorescence cells. On half of the slide will be fluorescing intensely. The line closest to the glowing cells is the edge of the electroporated area.

Any fluorescence beyond that is due to the transfer of lucer yellow through gap junctions. For example, this image of epithelial cells show cells that have been electro on the left side of the image, transferring Lucifer yellow to neighboring non electroporated cells, creating a gradient of glowing cells from the edge of the conductive coating toward the right. This image shows the same frame of cells under phase contrast.

Note that you can see the X line very clearly. A quantitative measure of how much gap junctional transfer has occurred can be done manually by working with the fluorescent and phase contrast images of electroporated cells. The stars along the edge of the conductive area are considered donor cells.

They have been electroporated and taken up loose for yellow. They pass luc for yellow to neighboring cells to the right, which pass it on. Still further in diminishing amounts.

The ratio of the dots to stars 41 to eight in this case can produce a number for comparing how extensive gap junctional transfer is in this case. 5.1 is the gap junction index. An alternative to manually marking and counting cells is to use image analysis software to measure the relative amounts of glowing along the non-elected Side of an electroporated cell border.

These cells do not have gap junctions. Here is the right edge of the electroporated area. Note, there is no gradient of fluorescence, which indicates the absence of gap junctions under phase contrast.

You can clearly see all the cells. Note That the X line is also clearly visible. In this example.

All of the cells in the chamber Were stimulated with epidermal growth factor EGF, and 10 minutes later fixed and probed for phospho arc. Note that the stained cells on the right have not been electroporated, and the cells on the left up to this line have been electroporated. The non electroporated cells show brown staining with a phospho irk antibody and immunoperoxidase.

The electroporated cells show no staining as the electroporated phospho peptide has blocked the SSH two domain of grab two and inhibited irk phosphorylation of EGF. Note the region of three to four cell length to the right of the line where cells were not electro yet do not show staining. These are cells that have acquired blocking peptide from their electroporated neighbors via gap junctions.

This is the most powerful demonstration possible that the inhibition of the signal must be due to the peptide and cannot be an artifact of electroporation. Since these cells are not electroporated, this is the same frame shown under phase contrast. Note the absence of any change in Morphology after electroporation.

With some patients gap junctional communication can be quantitative precisely In a large variety of cultured cells. The introduction of peptides to interrupt specific signaling pathways is a powerful approach to assess the relevance in live cells of protein, protein interactions that have been identified with purified proteins. This can Be the first important step in the development of peptid dome drugs.