We thank Lowell Cochran for expert videography assistance. The financial assistance of the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Breast Cancer Research Alliance, the Ontario Centers of Excellence, the Breast Cancer Action Kingston and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. SG was the recipient of an NSERC studentship. MG was supported by a postdoctoral fellowship from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#8217;s University.

1. Plating the Cells in the Electroporation Chambers
<ol>
	<li>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.</li>
	<li>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).</li>
	<li>When the cells have formed a confluent layer they are ready for GJIC examination.</li>
</ol>
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.
<ol>
	<li>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 4oC.</li>
	<li>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).</li>
	<li>With an Eppendorf pipettor, pipette the dye solution to the cells (400 &#956;l for the whole chamber), at the edge of the chamber, being careful not to touch the cell layer.</li>
	<li>Place the chamber in the holder supplied with the electroporator and apply a pulse of the appropriate strength (see Discussion).</li>
	<li>Carefully aspirate some of the LY solution. It can be reused a second time for less important experiments.</li>
	<li>Add Calcium-free DMEM containing 10% dialysed serum.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Microscopic Examination
<ol>
	<li>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.</li>
	<li>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).</li>
</ol>
4. Quantitation of Intercellular Communication
<ol>
	<li>Photograph the cells with a 20x objective under fluorescence and phase contrast (Figure 3).</li>
	<li>Identify and mark with a star the electroporated cells at the border with the non-electroporated area (Figure 2, arrow, electroporation edge).</li>
	<li>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).</li>
	<li>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.</li>
</ol>

<ol>
	<li>Raptis, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+Adherent+Cells+in+Situ+for+the+Study+of+Signal+Transduction+and+Gap+Junctional+Communication.">Electroporation of Adherent Cells in Situ for the Study of Signal Transduction and Gap Junctional Communication.</a> Electroporation protocols. , 167-183 (2008).</li><li>Giorgetti-Peraldi, S., Ottinger, E., Wolf, G., Ye, B., Burke, T. R., Shoelson, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+effects+of+phosphotyrosine-binding+domain+inhibitors+on+insulin+receptor+signalling+and+trafficking.">Cellular effects of phosphotyrosine-binding domain inhibitors on insulin receptor signalling and trafficking.</a> Mol Cell Biol. 17, 1180-1188 (1997).</li><li>Boccaccio, C., Ando, M., Tamagnone, L., Bardelli, A., Michielli, P., Battistini, C., Comoglio, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+epithelial+tubules+by+growth+factor+HGF+depends+on+the+STAT+pathway.">Induction of epithelial tubules by growth factor HGF depends on the STAT pathway.</a> Nature. 391, 285-288 (1998).</li><li>Bardelli, A., Longati, P., Gramaglia, D., Basilico, C., Tamagnone, L., Giordano, S., Ballinari, D., Michieli, P., Comoglio, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncoupling+signal+transducers+from+oncogenic+MET+mutants+abrogates+cell+transformation+and+inhibits+invasive+growth.">Uncoupling signal transducers from oncogenic MET mutants abrogates cell transformation and inhibits invasive growth.</a> Proc Nat Acad Sci USA. 95, 14379-14383 (1998).</li><li>Gambarotta, G., Boccaccio, C., Giordano, S., Ando, M., Stella, M. C., Comoglio, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ets+up-regulates+met+transcription.">Ets up-regulates met transcription.</a> Oncogene. 13, 1911-1917 (1996).</li><li>Arulanandam, R., Vultur, A., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfection+techniques+affecting+Stat3+activity+levels.">Transfection techniques affecting Stat3 activity levels.</a> Anal Biochem. 338 (1), 83-89 (2005).</li><li>Boussiotis, V. A., Freeman, G. J., Berezovskaya, A., Barber, D. L., Nadler, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+human+T+cell+anergy:+blocking+of+IL-2+gene+transcription+by+activated+Rap1.">Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1.</a> Science. 278 (5335), 124-128 (1997).</li><li>Brownell, H. L., Firth, K. L., Kawauchi, K., Delovitch, T. L., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+the+study+of+Ras+activation;+electroporation+of+[alpha-32]GTP.">A novel technique for the study of Ras activation; electroporation of [alpha-32]GTP.</a> DNA Cell Biol. 16, 103-110 (1997).</li><li>Tomai, E., Vultur, A., Balboa, V., Hsu, T., Brownell, H. L., Firth, K. L., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+electroporation+of+radioactive+compounds+into+adherent+cells.">In situ electroporation of radioactive compounds into adherent cells.</a> DNA Cell Biol. 22, 339-346 (2003).</li><li>Raptis, L., Vultur, A., Tomai, E., Brownell, H. L., Firth, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+electroporation+of+radioactive+nucleotides:+assessment+of+Ras+activity+and+32P-labelling+of+cellular+proteins.">In situ electroporation of radioactive nucleotides: assessment of Ras activity and 32P-labelling of cellular proteins.</a> Cell Biology A Laboratory Handbook, Celis, Ed. 43, 329-339 (2006).</li><li>Nakashima, N., Rose, D., Xiao, S., Egawa, K., Martin, S., Haruta, T., Saltiel, A. R., Olefsky, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+role+of+crk+II+in+actin+cytoskeleton+organization+and+mitogenesis.">The functional role of crk II in actin cytoskeleton organization and mitogenesis.</a> J Biol Chem. 274, 3001-3008 (1999).</li><li>Raptis, L., Firth, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+adherent+cells+in+situ.">Electroporation of adherent cells in situ.</a> DNA Cell Biol. 9, 615-621 (1990).</li><li>Marais, R., Spooner, R. A., Stribbling, S. M., Light, Y., Martin, J., Springer, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cell+surface+tethered+enzyme+improves+efficiency+in+gene-directed+enzyme+prodrug+therapy.">A cell surface tethered enzyme improves efficiency in gene-directed enzyme prodrug therapy.</a> Nat Biotechnol. 15, 1373-1377 (1997).</li><li>Nielsen, M. S., Nygaard, A. L., Sorgen, P. L., Verma, V., Delmar, M., Holstein-Rathlou, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctions.">Gap junctions.</a> Compr Physiol. 2 (3), 1981-2035 (2012).</li><li>Geletu, M., Trotman-Grant, A., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mind+the+gap;+regulation+of+gap+junctional,+intercellular+communication+by+the+SRC+oncogene+product+and+its+effectors.">Mind the gap; regulation of gap junctional, intercellular communication by the SRC oncogene product and its effectors.</a> Anticancer Res. 32 (10), 4245-4250 (2012).</li><li>Vinken, M., Vanhaecke, T., Papeleu, P., Snykers, S., Henkens, T., Rogiers, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexins+and+their+channels+in+cell+growth+and+cell+death.">Connexins and their channels in cell growth and cell death.</a> Cell Signal. 18 (5), 592-600 (2006).</li><li>Fouly, M. H., Trosko, J. E., Chang, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scrape-loading+and+dye+transfer:+A+rapid+and+simple+technique+to+study+gap+junctional+intercellular+communication.">Scrape-loading and dye transfer: A rapid and simple technique to study gap junctional intercellular communication.</a> Exp Cell Res. 168, 430-442 (1987).</li><li>Raptis, L., Brownell, H. L., Firth, K. L., MacKenzie, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+the+study+of+intercellular,+junctional+communication;+electroporation+of+adherent+cells+on+a+partly+conductive+slide.">A novel technique for the study of intercellular, junctional communication; electroporation of adherent cells on a partly conductive slide.</a> DNA &amp; Cell Biol. 13, 963-975 (1994).</li><li>Anagnostopoulou, A., Cao, J., Vultur, A., Firth, K. L., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+gap+junctional,+intercellular+communication+by+in+situ+electroporation+on+two+co-planar+indium-tin+oxide+electrodes.">Examination of gap junctional, intercellular communication by in situ electroporation on two co-planar indium-tin oxide electrodes.</a> Mol Oncol. 1, 226-231 (2007).</li><li>Tomai, E., Brownell, H. L., Tufescu, T., Reid, K., Raptis, S., Campling, B. G., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+assay+for+intercellular,+junctional+communication+in+cultured+human+lung+carcinoma+cells.">A functional assay for intercellular, junctional communication in cultured human lung carcinoma cells.</a> Lab Invest. 78, 639-640 (1998).</li><li>Tomai, E., Brownell, H. L., Tufescu, T., Reid, K., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gap+junctional+communication+in+lung+carcinoma+cells.">Gap junctional communication in lung carcinoma cells.</a> Lung Cancer. 23, 223-231 (1999).</li><li>Geletu, M., Chaize, C., Arulanandam, R., Vultur, A., Kowolik, C., Anagnostopoulou, A., Jove, R., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat3+activity+is+required+for+gap+junctional+permeability+in+normal+epithelial+cells+and+fibroblasts.">Stat3 activity is required for gap junctional permeability in normal epithelial cells and fibroblasts.</a> DNA Cell Biol. 28, 319-327 (2009).</li><li>Geletu, M., Arulanandam, R., Greer, S., Trotman-Grant, A., Tomai, E., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat3+is+a+positive+regulator+of+gap+junctional+intercellular+communication+in+cultured,+human+lung+carcinoma+cells.">Stat3 is a positive regulator of gap junctional intercellular communication in cultured, human lung carcinoma cells.</a> BMC Cancer. 12, 605 (2012).</li><li>Brownell, H. L., Narsimhan, R., Corbley, M. J., Mann, V. M., Whitfield, J. F., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ras+is+involved+in+gap+junction+closure+in+mouse+fibroblasts+or+preadipocytes+but+not+in+differentiated+adipocytes.">Ras is involved in gap junction closure in mouse fibroblasts or preadipocytes but not in differentiated adipocytes.</a> DNA &amp; Cell Biol. 15, 443-451 (1996).</li><li>Weber, P. A., Chang, H. C., Spaeth, K. E., Nitsche, J. M., Nicholson, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+permeability+of+gap+junction+channels+to+probes+of+different+size+is+dependent+on+connexin+composition+and+permeant-pore+affinities.">The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities.</a> Biophys J. 87 (2), 958-973 (2004).</li><li>Graham, F. L., vander Eb, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+technique+for+the+assay+of+infectivity+of+human+Adenovirus+5+DNA.">A new technique for the assay of infectivity of human Adenovirus 5 DNA.</a> Virology. 52, 456-467 (1973).</li><li>Arulanandam, R., Vultur, A., Cao, J., Carefoot, E., Truesdell, P., Elliott, B., Larue, L., Feracci, H., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-cadherin+engagement+promotes+survival+via+Rac/Cdc42+and+Stat3.">Cadherin-cadherin engagement promotes survival via Rac/Cdc42 and Stat3.</a> Mol Cancer Res 7. , 1310-1327 (2009).</li><li>Williams, E. J., Dunican, D. J., Green, P. J., Howell, F. V., Derossi, D., Walsh, F. S., Doherty, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+inhibition+of+growth+factor-stimulated+mitogenesis+by+a+cell-permeable+Grb2-binding+peptide.">Selective inhibition of growth factor-stimulated mitogenesis by a cell-permeable Grb2-binding peptide.</a> J. Biol. Chem. 272, 22349-22354 (1997).</li><li>Raptis, L., Brownell, H. L., Vultur, A. M., Ross, G., Tremblay, E., Elliott, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+inhibition+of+Growth+Factor-stimulated+ERK1/2+activation+in+intact+cells+by+electroporation+of+a+Grb2-SH2+binding+peptide.">Specific inhibition of Growth Factor-stimulated ERK1/2 activation in intact cells by electroporation of a Grb2-SH2 binding peptide.</a> Cell Growth Differ. 11, 293-303 (2000).</li><li>Meda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+the+molecular+permeability+of+connexin+channels.">Assaying the molecular permeability of connexin channels.</a> Methods Mol Biol. 154, 201-224 (2001).</li><li>Hofgaard, J. P., Mollerup, S., Holstein-Rathlou, N. H., Nielsen, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+gap+junctional+intercellular+communication+based+on+digital+image+analysis.">Quantification of gap junctional intercellular communication based on digital image analysis.</a> Am J Physiol Regul Integr Comp Physiol. 297 (2), R243-R247 (2009).</li><li>Raptis, L., Lamfrom, H., Benjamin, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+cellular+phenotype+and+expression+of+polyomavirus+middle+T+antigen+in+rat+fibroblasts.">Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts.</a> Mol Cell Biol. 5, 2476-2486 (1985).</li><li>Raptis, L., Marcellus, R. C., Whitfield, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+membrane-associated+protein+kinase+C+activity+correlates+to+tumorigenicity+but+not+anchorage-independence+in+a+clone+of+mouse+NIH+3T3+cells.">High membrane-associated protein kinase C activity correlates to tumorigenicity but not anchorage-independence in a clone of mouse NIH 3T3 cells.</a> Exp Cell Res. 207, 152-154 (1993).</li><li>Vultur, A., Cao, J., Arulanandam, R., Turkson, J., Jove, R., Greer, P., Craig, A., Elliott, B. E., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+to+cell+adhesion+modulates+Stat3+activity+in+normal+and+breast+carcinoma+cells.">Cell to cell adhesion modulates Stat3 activity in normal and breast carcinoma cells.</a> Oncogene. 23, 2600-2616 (2004).</li><li>Vultur, A., Arulanandam, R., Turkson, J., Niu, G., Jove, R., Raptis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stat3+is+required+for+full+neoplastic+transformation+by+the+Simian+Virus+40+Large+Tumor+antigen.">Stat3 is required for full neoplastic transformation by the Simian Virus 40 Large Tumor antigen.</a> Mol Biol Cell. 16, 3832-3846 (2005).</li></ol>

The corresponding author is the inventor in a patent held by Queen&#8217;s University, which has been licensed to Cell Projects Inc. The company or the University had no influence upon the contents of the paper.

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...

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 peptides2-4, oligonucleotides, such as antisense RNA, double-stranded DNA decoy oligonucleotides to inhibit transcription factor binding, or siRNA3,5,6, radioactive nucleotides7-10, 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, 800-1,000&#197;, 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 GJIC15,16. 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 p38hog 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 specimens20-23. 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 stage19,24.

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			<td height="42" style="height:42px;width:179px;">Name of Material/ Equipment</td>
			<td style="width:428px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:179px;">DMEM</td>
			<td style="width:428px;">Cellgro http://www.cellgro.com/</td>
			<td style="width:119px;">50-013-PB</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">DMEM without Calcium</td>
			<td style="width:428px;">Hyclone (Thermo scientific: http://www.thermoscientific.com)</td>
			<td style="width:119px;">SH30319-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Donor Calf Serum&#160;</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# B15-008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Fetal Bovine Serum</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# A15-751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Electroporation apparatus</td>
			<td style="width:428px;">Cell Projects Ltd UK: http://www.cellprojects.com/</td>
			<td style="width:119px;">ACE-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-04-CC</td>
			<td>4-wells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-08-CC</td>
			<td>8-wells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Lucifer Yellow</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-25-LY</td>
			<td>High purity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CelTak</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354240</td>
			<td>Cell and tissue adhesive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Fibronectin</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Collagen</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Poly-Lysine</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">P8920</td>
			<td></td>
		</tr>
	</tbody>
</table>

a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide

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.&#160; 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&#160;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.

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...

Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

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

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.

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

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JoVE Journal

Biology

9.8K Views.  Queen's University. 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.