title: Resveratrol is not compatible with a Fura-2-based assay for measuring intracellular Ca²⁺ signaling.
abstract: Fura-2 is a commonly used fluorescent Ca(2+) dye that allows an accurate determination of cytosolic Ca(2+) levels by measuring the emission obtained at 510 nm after alternating excitation at 340 nm and 380 nm (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 [Ca(2+)]. 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 340 nm and a strong emission at 510 nm upon excitation at 340 nm (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 [Ca(2+)]. 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 Ca(2+) signaling but that long-time effects can be assessed, provided that the resveratrol is carefully removed by appropriate wash steps.
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Campus Gasthuisberg O/N-I box 802, Herestraat 49, BE-3000 Leuven, Belgium.
authors.forename: Ram Chandra
authors.initials: RC
authors.lastname: Paudel
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Campus Gasthuisberg O/N-I box 802, Herestraat 49, BE-3000 Leuven, Belgium.
authors.forename: Santeri
authors.initials: S
authors.lastname: Kiviluoto
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Campus Gasthuisberg O/N-I box 802, Herestraat 49, BE-3000 Leuven, Belgium.
authors.forename: Jan B
authors.initials: JB
authors.lastname: Parys
authors.affiliation: KU Leuven, Laboratory of Molecular and Cellular Signaling, Department of Cellular and Molecular Medicine, Campus Gasthuisberg O/N-I box 802, Herestraat 49, BE-3000 Leuven, Belgium. Electronic address: geert.bultynck@med.kuleuven.be.
authors.forename: Geert
authors.initials: G
authors.lastname: Bultynck
mesh_terms: D020013:Calcium Signaling; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000737:chemistry*; D000077185:Resveratrol; D013267:Stilbenes / Q000494:pharmacology*
chemical_list: D005456:Fluorescent Dyes; D013267:Stilbenes; D000077185:Resveratrol; D016257:Fura-2
keywords: Ca(2+); Fluorescence; Fura-2-AM; HeLa cells; Interference; Resveratrol