Fluo-4 is a visible light-excitable calcium indicator derived from its predecessor, Fluo-3. Like Fluo-3, Fluo-4 is a non-fluorescent indicator whose intensity increases ~100 fold upon binding Ca²⁺. Additionally, Fluo-4 is an analog of Fluo-3 exhibiting minor structural modifications attributing to a brighter and more photostable indicator. By replacing two chlorine substituents with fluorines, Fluo-4 displays as wavelength shift of ~12 nm towards the blue spectrum. This establishes a new maximum absorption wavelength at 495 nm making Fluo-4 more efficiently excitable by the 488 nm argon laser line. This attributes to a brighter fluorescence signal at lower dye concentrations making Fluo-4 less phototoxic compared to Fluo-3. Fluo-4 has a slightly higher Ca²⁺ binding affinity than Fluo-3, with a K_d value of 345 nM. For these reasons, Fluo-4 is better alternative for use with confocal microscopy than Fluo-3.

Fluo-4 is available as a membrane-permeant acetoxymethyl (AM) ester or as a membrane-impermeant salt. As an AM ester, the increased hydrophobicity of Fluo-4, AM allows it to readily penetrate intact membranes of living cells, and hydrolysis by non-specific intracellular esterases activates Fluo-4'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-4 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.

Screen Quest™ calcium flux assays are the preferred method in cell-based high throughput screening (HTS) to determine agonist-stimulated and antagonist-inhibited signaling through G protein coupled receptors (GPCRs). To date approximately one third of drugs available on the market are designed to target GPCRs. The Screen Quest™ Fluo 4 No Wash Calcium Assay offers a convenient no-wash, homogeneous fluorescence-based assay for monitoring GPCRs and calcium channels, and to detect intracellular calcium mobilization across a broad spectrum of biological targets. The assay can be performed in a convenient 96-well or 384-well microtiter-plate format and can be easily adapted for automation. Screen Quest™ Calcium Assays are optimized and validated for the following instruments: FDSS^®, FLIPR^®, ViewLux™, NOVOStar, ArrayScan™, FlexStation^® and IN Cell Analyzer.

In past comparative studies, cells loaded with Fluo-4 have displayed favorably large dynamic ranges sensitive at detecting elementary and global Ca²⁺ fluxes. However, Fluo-4 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.