Concurrent measurements of the free cytosolic concentrations of H+ and Na+ ions with fluorescent indicators.

We report a method for the concurrent measurement of intracellular [Na+] ([Na+ ]i) and pH (pHi) in cells co-loaded with SBFI, a Na+-sensitive fluorophore, and either carboxy SNARF-1 or SNARF-5F, H+-sensitive fluorophores. With the optical filters specified, fluorescence emissions from SBFI and either SNARF derivative were sufficiently distinct to allow the accurate measurement of [Na+]i and pHi in rat hippocampal neurons. Neither the Na+ sensitivity of SBFI nor the pH sensitivities of carboxy SNARF-1 or SNARF-5F was affected by the presence of a SNARF derivative or SBFI, respectively. In addition, the calibration parameters obtained in neurons single-loaded with SBFI, carboxy SNARF-1 or SNARF-5F were not significantly influenced by the presence of a second fluorophore. In contrast to the established weak sensitivity of SBFI for protons, both SNARF derivatives appeared essentially insensitive to changes in [Na+]i. The utility of the technique was demonstrated in neurons co-loaded with SBFI and SNARF-5F, which was found to have a lower p Ka in situ than carboxy SNARF-1. There were no significant differences in the changes in [Na+]i and pHi observed in response either to intracellular acid loads imposed by the NH4+ prepulse technique or to transient periods of anoxia in neurons single-loaded with SBFI or SNARF-5F or co-loaded with both probes. The findings support the feasibility of using SBFI in conjunction with either carboxy SNARF-1 or SNARF-5F to concurrently and accurately measure [Na+]i and pHi.