Calcium-sensitive probes for the measurement of intracellular calcium: effects of buffer system and magnesium concentration.

The most commonly used calcium-sensitive probes for the measurement of [Ca2+]i are fluorescent probes such as fura-2 or luminescent probes like aequorin. There are inherent theoretical limitations and benefits associated with the use of either probe in the measurement of cytosolic calcium. Using a cultured human umbilical endothelial cell line, we have investigated the role of the buffer used, the pH and the magnesium concentration on [Ca2+]i measurements, as well as fura-2 loading conditions, using both fura-2 and aequorin. We report that the use of non-physiological buffers (HEPES) can lead to an elevation of [Ca2+]i whether measured with fura-2 or aequorin. In addition, using buffers with low magnesium concentrations (< 1 mM) or alkaline pH0 can result in an apparent elevation in the [Ca2+]i during the sustained phase of the cellular response. Taken together these data suggest that similar results in the measurements of intracellular calcium can be obtained irrespective of the probe utilized. In addition, our data demonstrate that the conditions for cellular studies must be selected with care, since numerous artefacts can be introduced into measurements of intracellular calcium by the use of non-physiological conditions.