Measuring cytosolic free calcium concentration in endothelial cells with indo-1: the pitfall of using the ratio of two fluorescence intensities recorded at different wavelengths

Indo-1 is a new fluorescent indicator of the intracellular free calcium concentration Cai++. Indo-1 may be used in a similar manner as its predecessor quin2 but offers the principal advantage that the Ca++ saturated form of the Ca++ chelator has a emission maximum different in wavelength from that of free indo-1 (400 nm versus 483 nm). Therefore, the ratio of the fluorescence intensity F emitted at 400 nm to that of the fluorescence intensity G emitted at 483 nm (or 500 nm) should be a measure of Cai++ independent of the total amount of intracellular dye. However, when indo-1 is loaded into endothelial cells (grown in culture on quartz coverslips) by incubation with the acetoxymethylester of indo-1 (indo-1/AM), the ester in not completely hydrolysed to indo-1 intracellularly. Fluorescence emitted by uncleaved indo-1/AM at wavelengths 483-500 nm interferes with the fluorescence of indo-1. Ester fluorescence is influenced not only by ester concentration but by the fluorescence emitted at 400 nm by Ca++ bound indo-1 as well. Therefore, the ratio F/G cannot reliably evaluate increases in Cai++ in endothelial cells although F/G would indicate a basal Cai++ constant with time. By contrast, the fluorescence F is a sensitive parameter of the intracellular concentration of Ca++ bound indo-1, in particular when the excitation wavelength is set to 332 nm. F was used to measure resting Cai++ in endothelial cells (132 +/- 22 nM; n = 22) and to demonstrate dose-dependent and reversible increases in Cai++ in response to stimulation with bradykinin.