A practical guide to the preparation of Ca(2+) buffers.

Calcium (Ca(2+)) is a critical regulator of an immense array of biological processes, and the intracellular [Ca(2+)] that regulates these processes is ~ 10,000 lower than the extracellular [Ca(2+)]. To study and understand these myriad Ca(2+)-dependent functions requires control and measurement of [Ca(2+)] in the nano- to micromolar range (where contaminating Ca(2+) is a significant problem). As with pH, it is often essential to use Ca(2+) buffers to control free [Ca(2+)] at the desired biologically relevant concentrations. Fortunately, there are numerous available Ca(2+) buffers with different affinities that make this practical. However, there are numerous caveats with respect to making these solutions appropriately with known Ca(2+) buffers. These include pH dependence, selectivity for Ca(2+) (e.g., vs. Mg(2+)), ionic strength and temperature dependence, and complex multiple equilibria that occur in physiologically relevant solutions. Here we discuss some basic principles of Ca(2+) buffering with respect to some of these caveats and provide practical tools (including freely downloadable computer programs) to help in the making and calibration of Ca(2+)-buffered solutions for a wide array of biological applications.