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 (Catalog Number 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

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 (Catalog Number 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.

This document (03.0070.150501r1) was last updated on Mon Oct 13 2025. All trademarks and registered trademarks mentioned herein are the property of their respective owners.

Fluo-8® Calcium Indicators

Key Features of Fluo-8^® AM: