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Fluo-8®, the Brightest Green Ca2+ Indicators
Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding Ca2+ have enabled researchers to investigate changes in intracellular free Ca2+ concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Fluo-3 and Fluo-4 are most commonly used among the 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® has been developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelength of maximum excitation @ ~490 nm and maximum emission @ ~520 nm. Fluo-8® AM only requires room temperature while Fluo-3 AM and Fluo-4 AM require 37°C cell loading. In addition, Fluo-8® is 2 times brighter than Fluo-4 AM, and 4 times brighter than Fluo-3 AM.
AAT Bioquest offers a set of our outstanding Fluo-8® reagents with different calcium binding affinities (Fluo-8®: Kd = 389 nM; Fluo-8H™: Kd = 232 nM; Fluo-8L™: Kd = 1.86 µM; Fluo-8FF™: Kd = 10 µM). We also offer versatile packing sizes to meet your special needs, e.g., 1 mg, 10x50 µg or 20x50 µg.
Fig. 1
U2OS cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well 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 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.
U2OS cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well 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 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 (03.0006.120501r1) was last updated on Mon Oct 13 2025. All trademarks and registered trademarks mentioned herein are the property of their respective owners.
Fluo-8®, the Brightest Green Ca2+ Indicators
Fig. 1
U2OS cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well 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 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.
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