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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.
Document: 03.0006.120501r1
Last updated Tue Sep 09 2025
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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