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Fluo-8®, AM
Calcium measurements are 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 AM and Fluo-4 AM are most commonly used among the visible light-excitable calcium indicators for live-cell calcium imaging. 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 are developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelengths of Ex/Em = ≤490/≤520 nm. Fluo-8® AM can be loaded into cells at room temperature, while Fluo-3 AM and Fluo-4 AM require 37°C for cell loading. In addition, Fluo-8® AM is two times brighter than Fluo-4 AM and four 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, 20x50 µg, and HTS packages) with no additional packaging charge.
U2OS 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, respectively, 100 µL of Fluo-3 AM, Fluo-4 AM and Fluo-8® AM in HHBS at a concentration of 4 uM in a 37 °C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.
U2OS 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, respectively, 100 µL of Fluo-3 AM, Fluo-4 AM and Fluo-8® AM in HHBS at a concentration of 4 uM in a 37 °C, 5% CO2 incubator for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.
protocol
Protocol
CatalogSize
Price
Quantity
210801 mg
Price
210815x50 ug
Price
2108210x50 ug
Price
2108320x50 ug
Price
 
Physical properties
Dissociation constant (Kd, nM)389
Molecular weight1046.93
SolventDMSO
Spectral properties
Correction factor (260 nm)1.076
Correction factor (280 nm)0.769
Extinction coefficient (cm -1 M -1)
23430
Excitation (nm)495
Emission (nm)516
Quantum yield
0.16
1
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200
CAS1345980-40-6
Instrument settings
Fluorescence microscope
ExcitationFITC
EmissionFITC
Recommended plateBlack wall/clear bottom
Fluorescence microplate reader
Excitation490
Emission525
Cutoff515
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling
Documents
Protocol
Safety Data Sheet (Catalog 21080)
Safety Data Sheet (Catalog 21081)
Safety Data Sheet (Catalog 21082)
Safety Data Sheet (Catalog 21083)
Certificate of Origin
Certificate of Analysis
Flyer: Calcium Indicators
Brochure: Physiological Probes & Assay Kits
Brochure: Calcium Detection Probes & Assay Kits
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Page updated on October 30, 2025