Abstract
Electroporation was initially developed for the introduction of DNA into cells which grow in suspension and was performed in a cuvette with two flat electrodes on opposite sides. Different configurations were subsequently developed for the electroporation of adherent cells in situ, while the cells were growing on nonconductive surfaces or a gold-coated, conductive support. We developed an assembly where the cells grow and are electroporated on optically transparent, electrically conductive indium-tin oxide (ITO). This material promotes excellent cell adhesion and growth, is inert and durable, and does not display spontaneous fluorescence, making the examination of the electroporated cells by fluorescence microscopy possible. The molecules to be electroporated are added to the cells and introduced through an electrical pulse delivered by an electrode placed on top of the cells. We describe several electrode and slide configurations which allow the electroporation of large numbers of cells for large-scale biochemical experiments or for the detection of changes in cell morphology and biochemical properties in situ, with control, nonelectroporated cells growing on the same type of ITO-coated surface, side by side with the electroporated ones. In a modified version, this technique can be adapted for the study of intercellular, junctional communication; the pulse is applied in the presence of a fluorescent dye, such as lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination. An assembly is also described for the electroporation of sensitive cells without the use of an upper electrode.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
1. Potter, H., Weir, L. and Leder, P. (1984) Enhancer-dependent expression of human kappa immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation. Proc. Nat. Acad. Sci. U.S.A. 81, 7161–7165.
2. Neumann, E., Schaefer-Ridder, M., Wang, Y., and Hofschneider, P. H. (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J. 7, 841–845.
3. Matsumura, T., Konishi, R., and Nagai, Y. (1982) Culture substrate dependence of mouse fibroblasts survival at 4°C. In Vitro. 18, 510–514.
4. Raptis, L. and Firth, K.L. (1990) Electroporation of adherent cells in situ. DNA Cell Biol. 9, 615–621.
5. Raptis, L. (2000) Specific inhibition of growth factor-stimulated ERK1/2 activation in intact cells by electroporation of a Grb2-SH2 binding peptide. Cell Growth Differ. 11, 293–303.
6. Raptis, L., Vultur, A., Brownell, H.L., and Firth, K.L. (2006) Dissecting pathways: in situ electroporation for the study of signal transduction and gap junctional communication. In: Celis, J.E. (ed.). Cell biology: a laboratory handbook. Academic, San Diego, CA, pp. 341–354.
7. Potter, H. and Cooke, S.W.F. (1992) Gene transfer into adherent cells growing on microbeads. In: Chang, D.C., Chassy, B.M., Saunders, J.A. and Sowers, A.E. (eds.). Guide to electroporation and electrofusion. Academic, San Diego, CA, pp. 201–208.
8. Kwee, S., Nielsen, H.V., and Celis, J.E. (1990) Electropermeabilization of human cultured cells grown in monolayers. Incorporation of monoclonal antibodies. Bioelectrochem. Bioenerg. 23, 65–80.
9. Zheng, Q. and Chang, D.C. (1991) High-efficiency gene transfection by in situ electroporation of cultured cells. Biochim. Biophys. Acta. 1088, 104–110.
10. Boitano, S., Dirksen, E.R., and Sanderson, M.J. (1992) Intercellular propagation of calcium waves mediated by inositol trisphosphate. Science. 258, 292–295.
11. Yang, T.A., Heiser, W.C., and Sedivy, J.M. (1995) Efficient in situ electroporation of mammalian cells grown on microporous membranes. Nucl. Acids. Res. 23, 2803–2810.
12. Jen, C.P., Wu, W.M., Li, M., and Lin, Y.C. (2004) Site-specific enhancement of gene transfection utilising an attracting electric field for DNA plasmids on the electroporation chip. J. microelectromech. sys. 13, 947–955.
13. Yamauchi, F., Kato, K., and Iwata, H. (2005) Layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA onto transparent indium-tin oxide electrodes for temporally and spatially specific gene transfer. Langmuir. 21, 8360–8367.
14. Giorgetti-Peraldi, S., Ottinger, E., Wolf, G., Ye, B., Burke, T.R., Jr., and Shoelson, S.E. (1997) Cellular effects of phosphotyrosine-binding domain inhibitors on insulin receptor signalling and trafficking. Mol. Cell. Biol. 17, 1180–1188.
15. Boccaccio, C., Ando, M., Tamagnone, L., et al. (1998) Induction of epithelial tubules by growth factor HGF depends on the STAT pathway. Nature. 391, 285–288.
16. Bardelli, A., Longati, P., Gramaglia, D., et al. (1998) Uncoupling signal transducers from oncogenic MET mutants abrogates cell transformation and inhibits invasive growth. Proc. Nat. Acad. Sci. U.S.A. 95, 14379–14383.
17. Gambarotta, G., Boccaccio, C., Giordano, C., Ando, M., Stella, M.C., and Comglio, M.C. (1996) Ets up-regulates met transcription. Oncogene. 13, 1911–1917.
18. Boussiotis, V.A., Freeman, G.J., Berezovskaya, A., Barber, D.L., and Nadler, L.M. (1997) Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1. Science. 278, 124–128.
19. Raptis, L., Vultur, A., Tomai, E., Brownell, H.L., and Firth, K.L. (2006) In situ electroporation of radioactive nucleotides: assessment of Ras activity and 32P-labelling of cellular proteins. In: Celis, J.E. (ed.). Cell biology: a laboratory handbook. Academic, San Diego, CA, pp. 329–339.
20. Nakashima, N., Ross, D.W., Xiao, S., et al. (1999) The functional role of crk II in actin cytoskeleton organization and mitogenesis. J. Biol. Chem. 274, 3001–3008.
21. Marais, R., Spooner, R.A., Stribbling, S.M., Light, Y., Martin, J., and Springer, C.J. (1997) A cell surface tethered enzyme improves efficiency in gene-directed enzyme prodrug therapy. Nat. Biotechnol. 15, 1373–1377.
22. Brownell, H.L., Lydon, N., Schaefer, E., Roberts, T.M., and Raptis, L. (1998) Inhibition of epidermal growth factor-mediated ERK1/2 activation by in situ electroporation of nonpermeant [(alkylamino)methyl]acrylophenone derivatives. DNA Cell Biol. 17, 265–274.
23. Chang, D.C. (1989) Cell poration and cell fusion using an oscillating electric field. Biophys. J. 56, 641–652.
24. Wegener, J., Keese, C.R., and Giaever, I. (2002) Recovery of adherent cells after in situ electroporation monitored electrically. Biotechniques. 33, 348–352.
25. Brownell, H.L., Firth, K.L., Kawauchi, K., Delovitch, T.L., and Raptis, L. (1997) A novel technique for the study of Ras activation: electroporation of [α32P]GTP. DNA Cell Biol. 16, 103–110.
26. Boussiotis, V.A., Freeman, G.J., Berezovskaya, A., Barber, D.L., and Nadler, L.M. (1997) Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1. Science. 278, 124–128.
27. Firth, K.L., Brownell, H.L., and Raptis, L. (1997) Improved procedure for electroporation of peptides into adherent cells in situ. Biotechniques. 23, 644–645.
28. Raptis, L., Brownell, H.L., Firth, K.L., and MacKenzie, L.W. (1994) A novel technique for the study of intercellular, junctional communication: electroporation of adherent cells on a partly conductive slide. DNA Cell Biol. 13, 963–975.
29. Raptis, L., Liu, S.K.W., Firth, K.L., Stiles, C.D., and Alberta, J.A. (1995) Electroporation of peptides into adherent cells in situ. Biotechniques. 18, 104–114.
30. Folkman, J. and Moscona, A. (1978) Role of cell shape in growth control. Nature. 273, 345–349.
31. Tomai, E., Brownell, H.L., Tufescu, T., et al. (1998) A functional assay for intercellular, junctional communication in cultured human lung carcinoma cells. Lab. Invest. 78, 639–640.
32. Brownell, H.L., Narsimhan, R.P., Corbley, M.J., Mann, V.M., Whitfield, J.J., and Raptis, L. (1996) Ras is involved in gap junction closure in mouse fibroblasts or preadipocytes but not in differentiated adipocytes. DNA & Cell Biol. 15, 443–451.
33. Tomai, E., Brownell, H.L., Tufescu, T., Reid, K., and Raptis, L. (1999) Gap junctional communication in lung carcinoma cells. Lung Cancer. 23, 223–231.
34. Xie, H.Q., Huang, R., and Hu, V.W. (1992) Intercellular communication through gap junctions is reduced in senescent cells. Biophys. J. 62, 45–47.
35. Raptis, L., Tomai, E., and Firth, K.L. (2000) Improved procedure for examination of gap junctional, intercellular communication by in situ electroporation on a partly conductive slide. Biotechniques 29, 222–226.
36. Anagnostopoulou, A., Vultur, A., Arulanandam, R., et al. (2006) Differential effects of Stat3 inhibition in sparse vs confluent normal and breast cancer cells. Cancer Lett. 242, 120–132.
37. Anagnostopoulou, A., Cao, J., Vultur, A., Firth, K., and Raptis L. (2007) Examination of gap junctional, intercellular communication by in situ electroporation on two co-planar indium-tin oxide electrodes. Molecular Oncology. 1, 226–231.
38. Raptis, L., Balabo, V., Hsu, T., et al. (2003) In situ electroporation of large numbers of cells using minimal volumes of material. Anal. Biochem. 317, 124–128.
39. Tomai, E., Vultur, A., Balboa, V., et al. (2003) In situ electroporation of radioactive compounds into adherent cells. DNA Cell Biol. 22, 339–346.
Acknowledgments
The financial assistance of the Canadian Institutes of Health Research, the Canadian Breast Cancer Research Alliance, the Natural Sciences and Engineering Research Council of Canada (NSERC), the Cancer Research Society Inc., and the Department of Defense Breast Cancer Research Program (BCRP-CDMRP) is gratefully acknowledged. We are grateful to Heather Brownell, Evangelia Tomai, Adina Vultur, Rozanne Arulanandam, and Aikaterini Anagnostopoulou for many helpful discussions.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press
About this protocol
Cite this protocol
Raptis, L., Firth, K.L. (2008). Electrode Assemblies Used for Electroporation of Cultured Cells. In: Li, S. (eds) Electroporation Protocols. Methods in Molecular Biology™, vol 423. Humana Press. https://doi.org/10.1007/978-1-59745-194-9_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-194-9_4
Publisher Name: Humana Press
Print ISBN: 978-1-58829-877-5
Online ISBN: 978-1-59745-194-9
eBook Packages: Springer Protocols