In measurements of the intracellular free calcium concentration ([Ca2+]) using either microelectrodes or fluorescent probes, calibration is normally carried out in EGTA calcium buffer solutions. In the first part of the article the general properties of calcium buffer solutions are discussed, the equations used to calculate the apparent calcium binding constant (Kapp) are derived, and the difficulties in the calculation are discussed. The effects of the purity of EGTA as well as the influence of calcium contamination on the buffer solutions are explained. Because of the difficulties in calculating Kapp, and the importance of EGTA purity and calcium contamination, it is suggested that it is easier to measure all three under the appropriate experimental conditions using the method of Bers (1982). In the second part a do-it-yourself guide to the preparation of EGTA calcium buffer solutions is given. An experimental example is provided using the Bers method to measure purity, contamination, and Kapp. It is concluded that unless all three factors are known it is not possible to prepare accurate EGTA calcium buffer solutions.

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4.5 and 3, respectively). At present, measurement of the Kapp and ligand purity in the appropriate solution at the desired pH and temperature would seem to be the best strategy to adopt rather than attempting to calculate the constant. Since no recognized international standard exists for [Mg2+] at the micromolar level, values in the literature for Kd, etc. in this range can only be regarded as approximate.

It is now generally accepted that the intracellular ionized magnesium concentration ([Mg2+]i) in muscle cells is around 1 mmol l−1; in heart muscle this means that from the total some 90‐95% is bound (see McGuigan et al. 1991a). Although binding will include sequestration by intracellular organelles, a large part of the binding is by ATP in the cytosol and an equilibrium exists in the cytosol between free ATP, ionized magnesium and Mg‐ATP. The extend of this equilibrium depends on the equilibrium constant of the reaction, which is a function of pH, temperature and ionic strength. This equilibrium constant is also important in the estimation of [Mg2+]i using 31P‐NMR. In this method the difference between the α and β peaks of ATP is measured and from this shift and the equilibrium constant between Mg2+ and ATP in the cytosol, the [Mg2+]i can be calculated (Vink, 1993).

The new fluorescent indicator, mag-fura-5, was evaluated for its ability to measure accurately physiological changes in cytosolic free magnesium. The apparent dissociation constants (Kd) of the fluorochrome for Mg2+, Mg2+/EGTA and Ca2+/EGTA solutions were 14.7 mM, 15.4 mM, and 1.8 mM respectively. The calculated difference in the fluorescence ratios and in the resulting pMg between the standards with low-Ca2+ or low H+ backgrounds and the corresponding samples with approximately physiological levels were not significant. In contrast, the changes due to an increased Ca2+ or H+ content were statistically significant, with mean pMg differences of 0.10 +/- 0.09 (P < 0.02) and 0.33 +/- 0.26 (P < 0.01) respectively. Repetitive measurements on 3 consecutive days yielded comparable data with differences not exceeding 4%. Because of the good reproducibility, it is suggested that the new fluorescent probe may be suitable for free cytosolic magnesium determinations in isolated cells.