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

Podsumowanie

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

Streszczenie

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.  Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

Wprowadzenie

The application of electrical current to a cell causes the formation of pores on the cell membrane, by a process termed electroporation. The pores allow the passage of a variety of nonpermeant molecules through the membrane. The electrical field can be controlled precisely, so that the pores formed are very small and reclose rapidly, with minimal disturbance to the cellular physiology. Interestingly, adherent cells can be grown on a glass slide coated with conductive and transparent indium-tin oxide (ITO) and electroporated in situ, that is on the surface where they grow, without being detached to be electroporated in suspension. Cells can grow very well on this surface, and as they are attached and extended, detailed microscopic observation is possible. Using this technique, small nonpermeant molecules can be introduced instantly and into essentially 100% of the cells, which makes this technique especially suitable for studies on the activation of components of a pathway following ligand stimulation of a receptor (reviewed in¹).

Electroporation has been used mostly for the introduction of DNA (also called electrotransfection). However, electroporation in situ can be valuable for the introduction of a large variety of molecules, such as peptides^2-4, oligonucleotides, such as antisense RNA, double-stranded DNA decoy oligonucleotides to inhibit transcription factor binding, or siRNA^3,5,6, radioactive nucleotides^7-10, proteins¹¹¹² or pro-drugs¹³. Following electroporation, cells can be lysed for biochemical analyses or fixed and stained with antibodies.

The conductive ITO coating is very thin, 800-1,000Å, so that cell growth is not disturbed by the difference in height of the two surfaces, as the cells grow across the edge of the conductive coating. This offers the advantage that non-electroporated cells can be grown side by side with electroporated ones, to serve as controls. The same approach can be used for the examination of gap junctional, intercellular communication (GJIC), as described in the Video.

Gap junctions are channels connecting the interiors of adjacent cells¹⁴. Gap junctional, intercellular communication plays an important role in tumor formation and metastasis, while oncogenes such as Src suppress GJIC^15,16. To examine GJIC a fluorescent dye such as Lucifer yellow (LY) is often introduced into cultured cells through microinjection or scrape-loading¹⁷ and the diffusion of the dye into neighboring cells is microscopically observed under fluorescence illumination. These techniques however invariably cause cell damage. We now describe a technique where cells are grown on a glass slide which is partly coated with ITO¹⁸. An electric pulse was applied in the presence of LY (or other dyes) causing its penetration into the cells growing on the conductive part of the slide, while dye migration to the adjacent, non-electroporated cells was microscopically observed through fluorescence illumination. To avoid disturbing sensitive cells that may tend to detach from the monolayer, an assembly was designed that did not require an electrode to be placed on top of the cells to apply the electrical current¹⁹. This approach offers the ability to quantitate GJIC in a large number of cells, without any detectable disturbance to cellular metabolism, as indicated by the absence of an effect on the length of the G1 phase following serum stimulation¹², increase in the levels of the fos protooncogene protein (Raptis, unpublished) or two kinases associated with cellular stress, the p38^hog or JNK/SAPK kinase¹. This approach made possible the examination of the link between levels of oncogene expression, transformation and GJIC¹⁸, as well as the effect of Src and Stat3 upon GJIC in a variety of cell types, including cells freshly cultured from lung tumor specimens^20-23. In addition, in situ electroporation with the setup described which lacks a top electrode has been successfully employed for the demonstration of gap junction closure upon adipocytic differentiation, although cellular attachment to the substrate is reduced at that stage^19,24.

Protokół

1. Plating the Cells in the Electroporation Chambers

1. In a laminar-flow hood, trypsinize the cells using sterile technique as usual.
   NOTE: It is very important to eliminate by centrifugation all traces of trypsin, because they may hinder spreading of the cells on the glass, hence the formation of adherens and gap junctions.
2. Pipette 1 ml of the cell suspension in the sterile electroporation chambers provided with the in situ electroporator (Figure 1), and place in a 37 ^oC, CO₂ incubator.
   NOTE: Cell adhesion can be improved by plating on fibronectin, collagen, poly-lysine or cell and tissue adhesive (see Materials Table).
3. When the cells have formed a confluent layer they are ready for GJIC examination.

2. Electroporation Procedure

Electroporation for GJIC examination can be conducted outside a laminar-flow hood, since it takes only a few minutes. If longer incubation times are required for a specific experiment, then it can be conducted entirely in a laminar-flow hood. In all cases, the chambers where the cells are grown must be sterile.

1. Prepare a 5 mg/ml Lucifer yellow solution: Dissolve 10 mg LY powder (provided with the electroporator) in 2 ml Calcium-free growth medium. For experiments that requires longer incubation times, filter-sterilize the solution and store at 4^oC.
2. Aspirate the growth medium and wash the cells with Calcium-free medium, being careful not to scratch the monolayer or dry the cells. If the cells are dried they usually have darker nuclei and pick up LY without electroporation. The effect is more pronounced in the middle of the slide which is more exposed to air drafts (Figure 4F).
3. With an Eppendorf pipettor, pipette the dye solution to the cells (400 μl for the whole chamber), at the edge of the chamber, being careful not to touch the cell layer.
4. Place the chamber in the holder supplied with the electroporator and apply a pulse of the appropriate strength (see Discussion).
5. Carefully aspirate some of the LY solution. It can be reused a second time for less important experiments.
6. Add Calcium-free DMEM containing 10% dialysed serum.
7. Incubate the cells for 3-5 min in the incubator to allow dye transfer through gap junctions.
   NOTE: The inclusion of dialysed serum at this point helps pore closure.
8. Wash the unincorporated dye with Calcium-free DMEM for live cell observation. Alternatively, cells can be fixed at this stage, through the addition of 4% formaldehyde to the well, then washed with PBS (phosphate-buffered saline). In all cases the washing must remove all background.
   NOTE: In preliminary experiments to determine the optimal conditions, if the voltage is too low, then it is possible to let the cells recover in the incubator for a few minutes and electroporate the same slide a second time, to save in the cost of slides.

3. Microscopic Examination

1. Observe the cells under fluorescence illumination, using an inverted microscope equipped with the appropriate filter for the dye being used. For Lucifer yellow, excitation is 423nm, emission 555nm, WBV filter for a Nikon IX70 microscope.
2. Eliminate meniscus effects by placing a glass cover-slip on top of the plastic well, after filling it to the top with liquid, to examine cell morphology under phase contrast. For better pictures, the well can be detached from the slide, and the coverslip placed on the frame. For fixed cells, the well can be filled with glycerol for microscopic observation, while for live cells it must be filled with growth medium.
   NOTE: Washing and photographing is easier if the cells are fixed with formaldehyde following the transfer of the dye (step 2.7 above). If the cells are not fixed, fluorescence is eliminated from the cells within approximately 60 min depending upon the cell type, voltage and amount of LY introduced while in fixed cells fluorescence is retained for several hours. However, fluorescence fades or dissipates after O/N incubation, even in fixed cells. For this reason, photographs must be taken soon after electroporation (Figure 2).

4. Quantitation of Intercellular Communication

1. Photograph the cells with a 20x objective under fluorescence and phase contrast (Figure 3).
2. Identify and mark with a star the electroporated cells at the border with the non-electroporated area (Figure 2, arrow, electroporation edge).
3. Identify and mark with a dot fluorescing cells on the non-electroporated side, where the dye has transferred through gap junctions (Figures 3A and 3B).
4. Divide the total number of fluorescing cells on the non-electroporated area by the number of electroporated cells along the edge (Figures 3A and 3B).
   NOTE: The transfer from at least 200 contiguous electroporated border cells is calculated for each experiment. The number obtained is the GJIC value.

Wyniki

Figure 2 shows rat liver epithelial T51B cells²², electroporated with LY and photographed under fluorescence (panel A and B), or phase-contrast (C) illumination, following fixation and washing. In panel A, the edge of the electroporated area is marked in red. The gradient of fluorescence to the right of the red line denotes transfer through gap junctions. In Figure 3A and 3B, the quantitation of gap junctional communication is shown: The cells at the edge of t...

Dyskusje

Critical steps in the protocol

Electroporated material
The purity of the material to be electroporated is very important. If the cells are very flat, then higher concentrations of the tracking dye must be used (up to 20 mg/ml for LY) and in such cases, purity is even more important than in cells with a more spherical shape¹. Besides LY a large variety of other dyes or nonpermeant molecules have been employed, such as a series of Alexa dyes to use as probes for c...

Materiały

Name	Company	Catalog Number	Comments
DMEM	Cellgro http://www.cellgro.com/	50-013-PB
DMEM without Calcium	Hyclone (Thermo scientific: http://www.thermoscientific.com)	SH30319-01
Donor Calf Serum	PAA: http://www.paa.com/	Cat.# B15-008
Fetal Bovine Serum	PAA: http://www.paa.com/	Cat.# A15-751
Electroporation apparatus	Cell Projects Ltd UK: http://www.cellprojects.com/	ACE-100
Chambers	Cell Projects Ltd UK	ACE-04-CC	4-wells
Chambers	Cell Projects Ltd UK	ACE-08-CC	8-wells
Lucifer Yellow	Cell Projects Ltd UK	ACE-25-LY	High purity
CelTak	BD Biosciences	354240	Cell and tissue adhesive
Fibronectin	Sigma Aldrich	F1141
Collagen	BD Biosciences	354236
Poly-Lysine	Sigma Aldrich	P8920