Calibration of free Zn²⁺ and Cu²⁺ concentrations in Tricine solution. (A) Fluorescence from Newport Green DCF (3 µM) was measured after the addition of varying concentrations of ZnCl₂ to HEPES- and Tricine-based solutions. HEPES and Tricine solutions used for these experiments only contained 1 mM HEPES and 10 mM Tricine, respectively. KCl was added to adjust the ionic strength to 0.15 M, pH 7.4, KOH. Apparent free concentrations of Zn²⁺ in Tricine solution (Tricine calculated free) were calculated using the Solver function of the MaxChelator program (Patton et al., 2004). (B) Calibration of free Cu²⁺ concentrations in Tricine solution using 3 µM Mag-Fura-2. The addition of CuCl₂ led to a dose-dependent quenching of the fluorescent signal. Solver was used to calculate the apparent free concentration of Cu²⁺ in Tricine solution (Tricine calculated free).