Self-referencing with ion-selective microelectrodes (ISMs) is a useful approach for monitoring near-real-time ion flux near single cells and across epithelia. While ISMs for H+, Ca2+, and K+ have been optimized for use with self-referencing, ISMs for two other primary inorganic ions, Na+ and Cl-, have not. In this study, we have characterized ISMs based on three Na+ ionophores (I, VI, and X) and one Cl- ionophore to assess their suitability for use with self-referencing. ISMs constructed with Na+ ionophore VI have short response times (approximately 100 ms) but possess nearly an order of magnitude less selectivity for Na+ over K+ than ISMs constructed with Na+ ionophore X. The Na+ ionophore X mixture was enhanced to give it a shorter response time while not compromising its selectivity. A Cl(-)-selective microelectrode was constructed and characterized with superior anionic selectivity compared with previously reported Cl- ISMs used with self-referencing. This Cl(-)-selective microelectrode, however, has a relatively slow response time (approximately 3 s), thus requiring changes to the self-referencing protocol. Self-referencing with these ISMs will enable near-real-time ion flux measurements for Na+ and Cl-
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