Background: Indium-tin-oxide (ITO) glass electrodes possess the properties of optical transparency and high electrical conductivity, which enables the electrical stimulation of cultured cells to be performed whilst also measuring the responses with fluorescent imaging techniques. However, the quantitative relationship between the intensity of the stimulating current and the cell response is unclear when using conventional methods that employ a separated configuration of counter and stimulation electrodes. New method: A quantitative electrical current stimulation device without the use of a counter electrode was fabricated. Results: Nerve growth factor (NGF)-induced differentiated PC12 cells were cultured on an ITO single glass electrode, and the Ca2+ response to electrical stimuli was measured using fluorescent Ca2+ imaging. ITO electrode devices with a width less than 0.1mm were found to evoke a Ca2+ response in the PC12 cells. Subsequent variation in the length of the device in the range of 2-10mm was found to have little influence on the efficiency of the electric stimulus. We found that the stimulation of the cells was dependent on the electrical current, when greater than 60μA, rather than on the Joule heat, regardless of the width and length of the conductive area. Comparison with existing method(s): Because of the cells directly in contact with the electrode, our device enables to stimulate the cells specifically, comparing with previous devices with the counter electrode. Conclusions: The ITO device without the use of a counter electrode is a useful tool for evaluating the quantitative neural excitability of cultured neurons.
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