AAT Bioquest

Fluo-8® Calcium Indicators

Carbachol dose responses were measured in HEK-293 cells with Fluo-8<sup>®</sup> AM
Carbachol dose responses were measured in HEK-293 cells with Fluo-8® AM (Cat# 21080) and Fluo-4 AM. HEK-293 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 100 µL of dye-loading solution containing Fluo-8® AM or Fluo-4 AM for 1 hour at room temperature. Carbachol (25 µL/well) was added by NOVOstar to achieve the final indicated concentrations. The fluorescence signals were measured at Ex/Em = 490/525 nm. The EC50 of carbachol measured with Fluo-8® AM is about 1.2 µM.
Fluo-3 and Fluo-4 were the most commonly used visible light-excitable calcium indicators. However, Fluo-3 AM and Fluo-4 AM are only moderately fluorescent in live cells upon esterase hydrolysis, and require harsh cell loading conditions to maximize their cellular calcium responses. Fluo-8® dyes have been developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelengths of maximum excitation @ ~490 nm and maximum emission @ ~520 nm. For cell loading, Fluo-8® AM only requires incubation at room temperature while Fluo-3 AM and Fluo-4 AM require incubation at 37 °C. In addition, Fluo-8® AM is 2 times brighter than Fluo-4 AM, and 4 times brighter than Fluo-3 AM in cells. AAT Bioquest offers a set of out-standing Fluo-8® reagents with different calcium binding affinities.

Key Features of Fluo-8® AM:

  • Faster, more readily loaded into cells than Fluo-3 AM and Fluo-4 AM. Only room temperature is required.
  • Brighter, much brighter than Fluo-3 AM and Fluo-4 AM in cells.
  • Convenient, almost identical spectra to those of Fluo-4 AM.

fluo-3 am
fluo-4 am
fluo-8 am

U2OS cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar 96-well black wall/clear bottom plate. The growth medium was removed, and the cells were incubated with 100 µL of 4 µM Fluo-3 AM, Fluo-4 AM and Fluo-8® AM (Cat# 21080) in HHBS at 37 °C for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope using FITC channel.


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