Interferences of resveratrol with fura-2-derived fluorescence in intracellular free-Ca 2+ concentration determinations

Resveratrol (3,4′,5-trihydroxy-trans-stilbene) is an antioxidant widely employed in cell physiology studies. It has been reported that it interferes with fura-2-derived fluorescence, making the employment of this dye nonviable. In this work, the interference of resveratrol with fura-2 determinations of intracellular free-Ca2+ concentration ([Ca2+]c) was examined. Solutions containing different concentrations of resveratrol, with or without fura-2, in the presence or in the absence of Ca2+, were analyzed by spectrofluorimetry. AR42J tumor cells were employed to study the influence of resveratrol on fura-2 fluorescence in living cells, by single cell fluorimetry. Resveratrol impaired the detection of fura-2-fluorescence emission (510 nm) at the 340, 360 and 380 nm excitation wavelengths. Resveratrol emitted fluorescence at 510 nm when lighted at all three excitation wavelengths. In addition, resveratrol emitted fluorescence at 380 nm when excited at 340 nm. Our observations suggest that the employment of the ratiometric properties of fura-2 to follow changes in [Ca2+]c in the presence of resveratrol is not viable. However, we think that the 380 nm excitation light could be employed. Results could be expressed as F0/F380, where F0 is the resting fluorescence and F380 is the value of fluoresce at a certain time point. We could follow changes in [Ca2+]c evoked by CCK-8, and we also detected Ca2+ mobilization by 100 µM resveratrol in AR42J cells. This investigation presents evidence demonstrating that resveratrol interferes with fura-2 fluorescence spectra. Nevertheless, a chance still exists if the 380 nm excitation wavelength is employed in the middle or low micromolar concentrations of resveratrol.