Resveratrol is not compatible with a Fura-2-based assay for measuring intracellular Ca2+ signaling

Abstract

Fura-2 is a commonly used fluorescent Ca2+ dye that allows an accurate determination of cytosolic Ca2+ levels by measuring the emission obtained at 510nm after alternating excitation at 340nm and 380nm (F340/F380 ratio). Previous studies, based on Fura-2 measurements, claimed that resveratrol, a polyphenol implicated in human health, triggered an acute rise in cytosolic [Ca2+]. In this report, we show that the spectral properties of resveratrol are not compatible with the fluorescent properties of Fura-2. Resveratrol displays a strong absorption of light at a wavelength of 340nm and a strong emission at 510nm upon excitation at 340nm (F340). As a consequence, the F340 values, but not the F380 values, are increased when incubating cells with resveratrol. Consequently the F340/F380 ratio values acutely increase upon addition of resveratrol, independently of changes in cytosolic [Ca2+]. Yet, we show that pretreating cells with resveratrol does not affect the F340/F380 ratios of Fura-2, provided that resveratrol is washed away before fluorescence measurement. These results indicate that Fura-2 is not suitable for assessing acute effects of resveratrol on Ca2+ signaling but that long-time effects can be assessed, provided that the resveratrol is carefully removed by appropriate wash steps.