AAT Bioquest


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

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.

Fluo3 vs Fluo4 am vs Fluo8

U2OS cells were seeded overnight at 40,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with, respectively, 100 µL of Fluo-3 AM, Fluo-4 AM and Fluo-8® AM in HHBS at a concentration of 4 µM in a 37°C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.

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.

Table 1. Fluo-8® green fluorescent calcium indicators for live cell calcium imaging.

Ex (nm)
Em (nm)
Unit Size
Cat No.
Fluo-8®, AM495516389 nM0.16∼200 fold1 mg21080
Fluo-8®, AM495516389 nM0.16∼200 fold10x50 µg21082
Fluo-8®, AM495516389 nM0.16∼200 fold20x50 µg21083
Fluo-8®, AM495516389 nM0.16∼200 fold5x50 µg21081
Fluo-8H™, AM495516232 nM0.16∼200 fold10x50 µg21091
Fluo-8H™, AM495516232 nM0.16∼200 fold1 mg21090
Fluo-8L™, AM4955161.9 µM0.16∼200 fold1 mg21096
Fluo-8L™, AM4955161.9 µM0.16∼200 fold10x50 µg21097
Fluo-8FF™, AM49551610 µM0.16∼200 fold10x50 µg21104
Fluo-8FF™, AM49551610 µM0.16∼200 fold1 mg21105
Fluo-8®, potassium salt495516389 nM0.16∼200 fold1 mg21087
Fluo-8®, potassium salt495516389 nM0.16∼200 fold10x50 µg21089
Fluo-8®, sodium salt495516389 nM0.16∼200 fold1 mg21086
Fluo-8®, sodium salt495516389 nM0.16∼200 fold10x50 µg21088
Fluo-8H™, sodium salt495516232 nM0.16∼200 fold10x50 µg21095
Fluo-8L™, sodium salt4955161.9 µM0.16∼200 fold10x50 µg21098
Fluo-8L™, sodium salt4955161.9 µM0.16∼200 fold1 mg21099
  1. Kd: Calcium dissociation constant.
  2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
  3. FCa/FFree: Increase in fluorescence intensity of calcium bound indicator relative to its calcium free indicator form.


Screening GPCR and Calcium Channel Targets

Screen Quest™ Fluo-8
Carbachol Dose Response. HEK-293 cells were seeded overnight at 40,000 cells/100 µL/well in a Costar black wall/clear bottom 96-well plate. The cells were incubated with 100 µL of dye-loading solution using the Screen Quest™ Fluo-8 No Wash calcium assay kit or Fluo-4 NW kit (according to the manufacturer's instructions) for 1 hour at room temperature. Carbachol (50µL/well) was added by NOVOstar (BMG Labtech) to achieve the final indicated concentrations.
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

Table 2. Screen Quest™ Fluo-8 No Wash Calcium Assays for screening GPCR and calcium channel targets.

Ex (nm)
Em (nm)
Unit Size
Cat No.
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit4955161 plates36314
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit49551610 plates36315
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit495516100 plates36316
Screen Quest™ Fluo-8 No Wash Calcium Assay Kit495516100 plates36316
Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*4955161 plates36307
Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*49551610 plates36308
Screen Quest™ Fluo-8 Medium Removal Calcium Assay Kit *Optimized for Difficult Cell Lines*495516100 plates36309


Tips to Improve Results

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.

Table 3. Probenecid and Pluronic® F-127 products for improving fluo-3 cell retention and aqueous solubility.

Unit Size
Cat No.
ReadiUse™ probenecid *25 mM stabilized aqueous solution*10x10 mL20062
ReadiUse™ probenecid, sodium salt *Water-soluble*10x77 mg20061
Probenecid *Cell culture tested* *CAS 57-66-9*10x72 mg20060
Pluronic® F-127 *20% solution in DMSO*10 mL20052
Pluronic® F-127 *Cell culture tested *10 g20050
Pluronic® F-127 *10% solution in water*10 mL20053