The fluorescent chelator Indo-1 can make simultaneous determinations of two intracellular ion concentrations, such as [Ca2+] and [Cd2+], or [Ca2+] and [Ba2+], in a normal cell suspension. The second ion can be detected even if its spectrum when bound to Indo-1 is same as for the calcium-bound or the ion-free Indo-1, as long as there is a change in height. This is because the mathematical analysis uses not only the spectral shape, but also takes into account increases in total signal intensity. For maximum accuracy, whole spectra were analyzed. When 3 mM [Ba2+] was added to a B cell line that had been stimulated with antiimmunoglobulin to open receptor operated calcium channels, there was a sudden drop in 400 nm Indo-1 fluorescence. Spectral analysis showed that this was due to a drop in intracellular [Ca2+], which was consistent with blockage of the receptor-operated calcium current by extracellular Ba2+. The conductance for Ba2+ was also observable as a slow rise in total fluorescence. There was also a slow increase in intracellular [Ca2+] as barium accumulated in the cell, which was tentatively attributed to blockage of the plasma membrane calcium pump by intracellular Ba2+.

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