A reevaluation of neuronal zinc measurements: artifacts associated with high intracellular dye concentration

The emergence of zinc as a potent neurotoxin has prompted the development of techniques suitable for the measurement of intracellular free zinc ([Zn(2+)](i)) in cultured cells. Accordingly, a new family of Zn(2+)-sensitive fluorophores has become available. Using ionophore-induced elevations of [Zn(2+)](i) in cultured neurons, we measured [Zn(2+)](i)-induced changes in the novel dyes FuraZin-1 and FluoZin-2 and compared them with the established [Zn(2+)](i)-sensitive fluorophores mag-fura-2 and Newport Green. All of these dyes effectively detected [Zn(2+)](i), and FuraZin-1, FluoZin-2, and Newport Green showed selectivity for [Zn(2+)](i) over [Ca(2+)](i) and [Mg(2+)](i). However, the dyes showed little difference in their apparent sensitivity to [Zn(2+)](i), even though their in vitro affinities for Zn(2+) varied from 20 nM to 3 microM. We show herein that this is a consequence of the relatively high concentrations of intracellular dye used in experiments of this nature. Thus, for the measurement of [Zn(2+)](i), the sensitivity of the reporting system is dominated by the intracellular dye concentration, whereas dye affinity is unimportant. We extend these findings to show that calibration of dye signal to ion concentration is critically dependent on precise measurement of intracellular dye concentration.