Abstract—This paper describes results involving the percentage cell lysis of SWLA-2 murine hybridomas produced by AC electric field pulses at 1kHz with pulse widths ranging from 1 ms to 1 second. Cells that had been exposed to the electric fields were cultured and replicate samples were examined at 48 hours to determine the number of viable cells. I. INTRODUCTION OEVENSPECK [1, 2] first described and patented the use of electric fields to kill micro-organisms in the early 1960s. Hamilton and Sale systematically analyzed the effects of pulsed—but not AC—electric fields on bacterial protoplasts, spheroplasts, and erythrocytes in 1967 and 1968 [3, 4]. This technology has proven useful for a wide range of applications in recent decades. In vivo delivery of molecules and genetic materials to eukaryotic cells has been carried out using electropermeabilization techniques with field strengths of less than lethal magnitudes [5]. These techniques have also been used for cell-cell fusion, such as the production of monoclonal antibodies [3, 6, 7]. As the fields can effectively kill bacteria and yeasts, pulsed electric fields of lethal magnitudes have proven useful for food preservation and the reduction of biofouling with microorganisms. [8-11]. Relevant to these research efforts is the ongoing investigation of the effect of the rapidly pulsed fields produced by Tasers on the human body.[12] The studies described in this paper, using AC pulses of varying pulse width and frequency, were performed to gain an understanding of the parameters for the lysing of eukaryotic cells using a pulse of AC electric field. Manuscript received April 8th, 2005. B. A. Oakley is with the School of Engineering and Computer Science, Oakland University, Rochester, MI 48309. (248-370-2435; fax: 248-370- 4633; e-mail: oakley@oakland.edu). D. M. Hanna is with the Computer Science and Engineering Department, Oakland University, Rochester, MI 48309 (e-mail: dmhanna@oakland.edu). S. Kandlikar is with the Department of Biological Sciences, Oakland University, Rochester, MI 48309 (e-mail: sskandli@oakland.edu). B. Gross is with the Computer Science and Engineering Department, Oakland University, Rochester, MI 48309 (e-mail: bagross@oakland.edu). G. A. Stryker is with the Department of Biological Sciences, Oakland University, Rochester, MI 48309 (e-mail: gstryker@oakland.edu). II. MATERIALS AND METHODS A. Cell line The cell line used in this experiment was the SWLA-2 murine hybridoma (HB12560), purchased from ATCC [13]. The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with L-glutamine (0.8 mM), sodium pyruvate (1 mM), non-essential amino acids (0.1 mM), Hepes buffer (20 mM), and 10% fetal calf serum. The cells were grown at 37°C in 5% CO 2 . B Preparation The hybridomas used in these experiments were prepared as follows prior to the experiments. The total number of cells was obtained by counting them in Neubauer's hemocytometer using Trypan blue stain 0.2%. Cells were centrifuged at 130 × g for 10 min. The supernatant was discarded and the pellet was re-suspended at 3 x 10 5 cells/ml. C. Electrical Equipment and Cuvettes Sinusoidal electric field pulses of varying pulse length (1 ms to 1 second) and frequency were delivered to the cell suspensions by a lab-built setup that was capable of providing approximately 500 W with an rms voltage maximum of approximately 90 V. Eppendorf electroporation cuvettes with a 1 mm gap width and 100 µl volume were used to expose cells to the electric field. The voltage from the lab-built setup was applied across the 1 mm gap width of the cuvette. D. Determination of percentage of viable cells For all of the experiments conducted, the cells inoculated into 3 replicate wells of a 96-well plate (30 µl cells/well) containing 170 µl of culture media. Each experiment was then repeated on a subsequent sample (cuvette) and added into 3 replicate wells of second 96 well plate. The number of viable cells was determined by counting the number of live cells in Neubauer’s hemocytometer with trypan blue staining. The cells were counted 48 hours after exposure to the fields. The cell counts were relative to control cells that had not been exposed to electric fields. III. RESULTS AND DISCUSSION Results at 1 kHz are shown in Table 2. Hamilton and Sale [3], who demonstrated that to affect microorganisms and produce permeabilization of a cell membrane, electric Cell Lysis in SWLA-2 Hybridomas due to 1 kHz AC Electric Fields Barbara A. Oakley, Senior Member, IEEE, Darrin M. Hanna, Member, IEEE, Sachin Kandlikar, Brooks Gross, Member, IEEE, Gabrielle A. Stryker D