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 benefit s associated with the use of either probe in the measurement of cytoso lic calcium. Using a cultured human umbilical endothelial cell line, w e have investigated the role of the buffer used, the pH and the magnes ium concentration on [Ca2+]i measurements, as well as fura-2 loading c onditions, using both fura-2 and aequorin. We report that the use of n on-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 pH(0) can result in an apparent elevation in the [Ca2+](i) during the sustained phase of the cellular response. Taken together these data suggest that simil ar results in the measurements of intracellular calcium can be obtaine d irrespective of the probe utilized. In addition, our data demonstrat e that the conditions for cellular studies must be selected with care, since numerous artefacts can be Introduced into measurements of intra cellular calcium by the use of non-physiological conditions. (C) 1995 Academic Press, Inc.