Fluo-3 AM


The Ca2+ indicator fluo-3 was developed by Tsien and colleagues for use with visible-light excitation sources in flow cytometry and confocal laser scanning microscopy. Fluo-3 imaging has revealed the spatial dynamics of many elementary processes in Ca2+ signaling. Since about 1996, fluo-3 has also been extensively used in cell-based high-throughput screening assays for drug discovery. Fluo-3 is essentially nonfluorescent unless bound to Ca2+ and exhibits a quantum yield at saturating Ca2+ of ~0.14. The intact acetoxymethyl ester derivative of fluo-3 AM is therefore also nonfluorescent, unlike the AM esters of fura-2 and indo-1. The green-fluorescent emission (~525 nm) of Ca2+-bound fluo-3 is conventionally detected using optical filter sets designed for fluorescein (FITC). As prepared by Minta, Kao and Tsien, fluo-3 was originally reported to undergo an ~40-fold increase in fluorescence upon binding Ca2+. Fluo-3 currently produced by Molecular Probes exhibits an at least 100-fold Ca2+-dependent fluorescence enhancement. In a careful study of the spectral properties of highly purified fluo-3, Harkins, Kurebayashi and Baylor characterized the effects of pH and viscosity on Ca2+ measurements with fluo-3 and demonstrated that binding of the indicator to proteins has a significant effect on its Kd for Ca2+. The fluorescence output of fluo-3 — the product of the molar absorptivity and the fluorescence quantum yield — may also vary significantly in different cellular environments.
Fluo-3 lacks a significant shift in emission or excitation wavelength upon binding to Ca2+, which precludes the use of ratiometric measurements (Loading and Calibration of Intracellular Ion Indicators). Simultaneous loading of cells with fluo-3 and our Fura Red indicator (see below), which exhibit reciprocal shifts in fluorescence intensity upon binding Ca2+, has enabled researchers to make ratiometric measurements of intracellular Ca2+ using confocal laser scanning microscopy or flow cytometry. For ratiometric measurements, fluo-3 can also be co-loaded into cells with a Ca2+-insensitive dye. For instance, carboxy SNARF-1 AM, acetate— a dye that can be excited at the same wavelengths as fluo-3 but can be detected at much longer wavelengths— can serve as the Ca2+-insensitive dye, provided that the pH within the cells remains constant during the experiment.Co-loading of fluo-3 and carboxy SNARF-1 also permits the simultaneous imaging of Ca2+ transients and intracellular pH in experiments in which the concentrations of both ions are changing. Fluo-3 and some other Ca2+ indicators are useful for two-photon laser scanning microscopy.
Fluo-3 is available as a cell-impermeant potassium salt or ammonium salt. The cell-permeant fluo-3 AM is available as a 1 mg vial, as a set of 20 vials each containing 50 µg, as a set of 10 vials each containing 50 µg of FluoroPure grade fluo-3 AM and as a 1 mM solution in DMSO. A set of 40 vials, each containing 1 mg of fluo-3 AM, is available at a discounted price to provide a larger quantity for high-throughput screening applications.