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.

SUMMARYIntracellular free magnesium ([Mg2+]

SUMMARYBecause of the reported presence of a Na+-Mg2' exchanger in guinea-pig but not in ferret myocardium, the Mg2+ extrusion mechanism in guinea-pig myocardium has been reinvestigated using Mg2+-and Na+-selective microelectrodes and the fluorochromes mag-fura- In vitro calibration of mag-fura-2 and -5 using the ratio method gave values for Kd (experimentally determined dissociation constant) of 22 2 + 2-7 (mean + S.D., n = 7) and 25-7+ 1 3 (n = 4) mmol/l respectively. Mag-fura-2 reacted to physiological concentrations of Ca2+ and mag-fura-5 to changes in pH. In isolated myocytes, Na+ removal gave an apparent increase of [Mg2+], with mag-fura-2 but not with mag-fura-5. However, when the pH, was altered with NH4Cl mag-fura-5 showed an apparent decrease in [Mg2+]i on application and an apparent increase on removal, with a time course similar to the pH, changes. It is concluded that Mg2+ extrusion in guinea-pig myocardium is not via a Na+-Mg2+ exchanger. The use of mag-fura-2 and -5 are limited in their application because of Ca2+ and H+ sensitivity respectively.

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