Intracellular Ca2+ measurements in living cells

Abstract

Intracellular Ca2+ acts as the second messenger in a variety of cells. Many cellular functions are tightly regulated by the intracellular Ca2+ concentrations ([Ca2+]i). Therefore, measurement of the [Ca2+]i is of critical importance. Calcium-sensitive dual excitations, indicators, such as fura-2, are now widely used to measure the [Ca2+]i in living cells. The development of techniques allowing the measurement of [Ca2+] i has contributed noticeably to our understanding of many cellular functions. Digital video microscopy, confocal laser scanning microscopy, and multiphoton microscopy allow accurate spatial analysis of [Ca2+] i at the subcellular level. Fura-2 has been used commonly to measure the [Ca2+]i because of the sensitivity and specificity of the method. It can be loaded into living cells with little disruption of the cellular functions. Fura-2 acetoxymethyl ester (AM) is a lipid-soluble derivative that is often used extracellularly because of its ability to pass through cell membranes, whereas fura-2 by itself cannot be introduced into the cells. For fura-2 measurements, measurements of the fluorescence intensities at two excitation wavelengths can be used to obtain an estimate of the [Ca 2+]i, independent of the dye concentration and the thickness of the cell membrane?. In this review, I summarize the advantages and pitfalls of using fura-2 and other standard Ca2+ indicators, methods used to measure the [Ca2+]i, including simultaneous measurements of cell movements and the changes in the [Ca2+] i, and procedures used for measuring the cellular and/or subcellular Ca2+ concentrations in living cells from the cochlea, such as the outer and inner hair cells.