Fluo-3 is one of the most commonly utilized green calcium indicators available. It was first developed by Tsien and colleagues in the late 1980s (Grynkiewicz et al. 1985). Since then, Fluo-3 and its analogs have been utilized in many Ca²⁺ imaging applications. Making considerable contributions to understanding the spatial dynamics of processes associated with Ca²⁺ signaling pathways.

Fluo-3 is a visible light-excitable, single-wavelength Ca²⁺ indicator with spectral characteristics similar to fluorescein. Fluo-3 is well excited by the 488 nm argon laser line and can be visualized using the FITC emission filter sets. It has a maximum absorption and emission wavelength at 506 nm and 526 nm, respectively. Compared to other Ca²⁺ indicators, Fluo-3 has a relatively lower affinity for Ca²⁺. It exhibits a calcium dissociation constant (K_d) of ~390 nM which can alleviate drawbacks associated with cytosolic buffering at resting Ca²⁺ levels of ~100 nM. In the Ca²⁺ free form, Fluo-3 is essentially non-fluorescent. Upon binding Ca²⁺, the fluorescence intensity of Fluo-3 increases ~100 fold and at saturating Ca²⁺ levels exhibits a quantum yield of ~0.15. In addition to being sensitive to temperature changes, Fluo-3 is also pH-sensitive and sensitive to protein binding. Fluo-3 has been successfully used in flow cytometry, confocal and fluorescence microscopy and fluorescence microplate-screening assays.

Fluo-3 is available as a membrane-permeant acetoxymethyl (AM) ester or as a membrane-impermeant salt. As an AM ester, the increased hydrophobicity of Fluo-3, AM allows it to readily penetrate intact membranes of living cells, and hydrolysis by non-specific intracellular esterases activates Fluo-3's calcium sensitive fluorescence. Cells may also be physically loaded with membrane-impermeant calcium indicators, such as salt derivatives and dextran conjugates. Common cell loading techniques include patch pipette, microinjection or pinocytic loading. Fluo-3 should be stored desiccated and protected from light at ≤ -20°. As received AM esters can typically be stored for at least six months, but beware they are susceptible to hydrolysis, particularly when stored in solution.

In past comparative studies, cells loaded with Fluo-3 have displayed favorably large dynamic ranges sensitive at detecting elementary and global Ca²⁺ fluxes. However, Fluo-3 requires incubation at 37°C resulting in significant indicator loss by the increase activity of anion transporters sensitive to the same temperature. To improve cellular retention reagents such as probenecid can be added to the dye working solution. Probenecid works be inhibiting organic anion transporters located in the cell membranes. Unfortunately, probenecid can be toxic to cells and while it inhibits certain anion transporters it may activate others. For example, the TRPV2 receptors of sensory ganglia have been shown to be activated by probenecid. On the other hand, probenecid seems to have an inhibitory effect on TAS2R receptors, or taste receptors. For many other serum-sensitive or drug-sensitive targets, the effects of probenecid could be largely unknown. This means that by using probenecid, a research risks the introduction of a completely unknown factor into an experimental design.

Noting this critical problem with existing calcium indicators, Calbryte™ 520 has been specifically designed for high performance without probenecid. Calbryte™ 520 is our upgrade for traditional green fluorescent indicators such as Fluo-3 and Fluo-4. This dye has an excitation maximum at 493 nm, which closely matches the standard 488 nm argon ion laser line. Also, with an emission maximum at 515 nm, this dye is compatible with the FITC filter sets found in most fluorescence instruments. Because Calbryte™ 520 has spectral values nearly identical to that of Fluo-3, no additional instrument setup is required. This allows for a seamless and convenient transition between products.