Calcium buffer solutions and how to make them: a do it yourself guide

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.