A practical guide to the preparation of Ca2+ buffers.

TLDR: Some basic principles of Ca(2+) buffering are discussed and practical tools are provided to help in the making and calibration ofCa(2+)-buffered solutions for a wide array of biological applications.

Abstract: Calcium (Ca2+) is a critical regulator of an immense array of biological processes, and the intracellular [Ca2+] that regulates these processes is ~ 10,000 lower than the extracellular [Ca2+]. To study and understand these myriad Ca2+-dependent functions requires control and measurement of [Ca2+] in the nano- to micromolar range (where contaminating Ca2+ is a significant problem). As with pH, it is often essential to use Ca2+ buffers to control free [Ca2+] at the desired biologically relevant concentrations. Fortunately, there are numerous available Ca2+ buffers with different affinities that make this practical. However, there are numerous caveats with respect to making these solutions appropriately with known Ca2+ buffers. These include pH dependence, selectivity for Ca2+ (e.g., vs. Mg2+), ionic strength and temperature dependence, and complex multiple equilibria that occur in physiologically relevant solutions. Here we discuss some basic principles of Ca2+ 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 Ca2+-buffered solutions for a wide array of biological applications.