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Fluo-8® Calcium Indicators
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.
Fig. 1
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.
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.
Fig. 2
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.
Document: 03.0070.150501r1
Last updated Tue Sep 09 2025
Fluo-8® Calcium Indicators
Key Features of Fluo-8® AM: