Notes & Tips In situ electroporation of large numbers of cells using minimal volumes of material Leda Raptis, a,b, * Valerie Balboa, a,b Tina Hsu, a,b Adina Vultur, b James Turkson, c Richard Jove, c and Kevin L. Firth d a Department of Microbiology and Immunology, QueenÕs University, Kingston, Ontario, Canada K7L3N6 b Department of Pathology, QueenÕs University, Kingston, Ontario, Canada, K7L3N6 c Moffitt Cancer Center, Tampa, FL, USA d Ask Science Products Inc. Kingston, Ontario, Canada Received 10 November 2002 Electroporation is a technique utilizing the ability of brief electric pulses to form transient pores through the cell membrane for the introduction of nonpermeant molecules into cultured cells (reviewed in [1]). We have developed a modified version for adherent cells, termed electroporation in situ, where the cells receive the pulse while they are growing on a glass surface coated with electrically conductive, optically transparent indium–tin oxide (ITO). 1 This material is inert and nontoxic to the cells, promotes excellent cell adhesion and growth, and permits direct visualization of the cells by phase-contrast or fluorescence microscopy. Careful control of the electric field intensity results in essentially 100% of the cells taking up the introduced material, without any detectable effect upon the physiology of the cell, pre- sumably because the pores reseal rapidly so that the cellular interior is restored to its original state [2,3]. However, electroporation of large numbers of cells is faced with two problems: (1) the high cost of the ma- terial electroporated and (2) the burning of the ITO coating under the high voltages required to electropo- rate the larger areas. In this communication we describe a crucial modification in the design of electrodes and slides where large numbers of cells can be effectively electroporated using a minimal volume of material. The results indicate that introduction of a decoy oligonu- cleotide with this assembly can inhibit Stat3-mediated transcription. Moreover, the possibility for upscaling made the demonstration of the inhibition of EGF- mediated Erk1/2 activation through electroporation of a Grb2–SH2 binding peptide in sparse NIH3T3 fibro- blasts possible. Cells are grown on ITO-coated slides placed in a petri dish to maintain sterility [4] (Fig. 1). The cell growth area is defined by a ‘‘window’’ formed with an electri- cally insulating frame made of Teflon. The pulse is transmitted through a stainless steel negative electrode placed above the cells, resting on the Teflon frame. Another stainless steel block serves as a positive contact bar. To obtain a uniform electric field intensity over the entire area below the negative electrode, despite the fact that the conductive coating exhibits a significant amount of electrical resistance (surface resistivity of >2 X/ square), the bottom surface of the negative electrode must be inclined relative to the glass surface, rising in the direction of the positive contact bar [4]. Extensive pre- vious experience indicated that uniform electroporation using slides with surface resistivity of 2 X/square re- quires an angle of 1.5°, which, for a cell growth area of 32  10 mm and a frame with a thickness of 0.279 mm, translates into approximately 140 ll of solution which is held in place by surface tension [4,5]. Problems in electroporating costly material The above assembly has been extensively employed for the introduction of peptides, oligonucleotides, and a large variety of other nonpermeant compounds [5–9]. Previous experience however, demonstrated that in most instances electroporation must be conducted under a Analytical Biochemistry 317 (2003) 124–128 www.elsevier.com/locate/yabio ANALYTICAL BIOCHEMISTRY * Corresponding author. Fax: 613-533-6796. E-mail address: raptisl@post.queensu.ca (L. Raptis). 1 Abbreviations used: ITO, indium–tin oxide; STAT, signal trans- ducer and activator of transcription; EGF, epidermal growth factor. 0003-2697/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-2697(03)00078-2