Fluo-8

Fluo-8^® is a novel green calcium indicator which incorporates the same fluorescein core, utilized in Fluo-3 and Fluo-4, to monitor Ca²⁺ concentration and flux in cells. This enables Fluo-8^® to retain spectral properties identical to Fluo-4 while improving the limitations plaguing Fluo-3 and Fluo-4. Minor structural modifications attributing to the inception of Fluo-8^®, has led to several enhancements associated with its employment, one being Fluo-8^®'s improved loading conditions. Compared to Fluo-3 and Fluo-4, that require indicator loading at a temperature of 37°C, Fluo-8^® can be loaded successfully at room temperature reducing the required loading time to just 20 minutes.

Fluo-8^® Indicators for Calcium Imaging

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Like the other Fluo-dyes, Fluo-8^® is non-fluorescent in the Ca²⁺ free form. Upon binding to Ca²⁺, Fluo-8^® displays a larger fluorescence intensity increase of ~200 fold. This is significantly two times brighter than Fluo-4 and four times brighter than Fluo-3. Fluo-8^® is less temperature dependent compared to other probes, which allows for more consistent and reproducible results either at room temperature or 37 °C. At these conditions, Fluo-8^® can be readily loaded into cells in 30 minutes or less. Fluo-8^® and its analogs are available in four distinct forms each expressing different calcium binding affinities. Fluo-8^® has a Kd of ~389 nM, Fluo-8™H has a Kd of ~232 nM, Fluo-8™L has a Kd of ~1.86 µM and Fluo-8™FF has a Kd of ~10 µM. Fluo-8^®L and Fluo-8™FF have significantly lower Ca²⁺ binding affinities, making them better suited for detecting intracellular Ca²⁺ levels in the micromolar range.

AM Ester and Salt Derivatives

Fluo-8^® is available as a membrane-permeant acetoxymethyl (AM) ester or as a membrane-impermeant salt. As an AM ester, the increased hydrophobicity of Fluo-8^®, AM allows it to readily penetrate intact membranes of living cells, and hydrolysis by non-specific intracellular esterases activates Fluo-8^®'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-8^® 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.

1. K_d: Calcium dissociation constant.
2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
3. F_Ca/F_Free: Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.

Screening GPCR and Calcium Channel Targets

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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-8 No Wash Calcium Assay offers a convenient no-wash, homogeneous fluorescence-based assay for detecting intracellular calcium mobilization across a broad spectrum of biological targets. The Screen Quest™ Fluo-8 No Wash Calcium Assay Kits includes an optimized assay method for monitoring GPCRs and calcium channels. Unlike Fluo-3 AM and Fluo-4 AM, both of which require 37°C for optimal cell loading, the Screen Quest™ Fluo-8 No Wash Calcium Assay requires a less temperature-dependent cell loading property, giving similar results either at room temperature or 37°C. This characteristic makes the Screen Quest™ Fluo-8 No Wash Calcium Assay Kits more robust for HTS applications. In addition, its ability to generate very large signals from weak and robust assay systems enables researchers to have multiple assay chemistries for different receptor and calcium channel 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.

Screen Quest™ Fluo-8 Key Features

• Optimized protocols validated for a broad range of microplate readers
• Superior signal intensity (high S/B ratios) and well-to-well uniformity
• Large assay window for assaying challenging cell lines and receptors
• High performance calcium flux assay for 1536 well-plates

Tips to Improve Results

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Cells loaded with Fluo-8^® have displayed favorably large dynamic ranges sensitive at detecting elementary and global Ca²⁺ fluxes. However, Fluo-8^® is susceptible to dye extrusion by organic anion transporters sensitive to 37°C. 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, therefor it should be used with discretion. To improve the aqueous solubility of AM esters, also add Pluronic^® F-127 to the Fluo-8 working solution. Pluronic^® F-127 is a nonionic surfactant that is 1005 active and relatively non-toxic to cells. At low temperatures it is soluble, however at room temperature Pluronic^® F-127 will gel.