Fluo-8 has become the most popular of many names used to market a specific version of the molecule patented and introduced to the world as Fluo-2 in 1988-1989 (US Patent 5049673, 1991). Fluo-8 is Fluo-2 Medium Affinity which replaces the Methyl group of the Fluo-2 High Affinity with a Hydrogen as described below.
Calcium measurement is critical for a wide range of biological investigations. The development of fluorescent probes that show spectral responses upon binding Ca2+ has enabled researchers to investigate changes in intracellular free Ca2+ concentrations using a variety of techniques including: fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers.
The Fluo-8 molecule( originally designated as Fluo-2 medium affinity in the 1991 patent), is one of the original Fluo series of Ca2+ indicators invented by Drs. Roger Tsien and Akwasi Minta in the 1980s. The Fluo-3 and Fluo-4 molecules have been the 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 AM hydrolysis, and depending on cell type and application, loading can be difficult to maximize cellular response.
Whereas Fluo-3 and Fluo-4 were commercialized, Fluo-8 was not. Subsequently, Fluo-8 has been found to be much brighter (1.5x) than Fluo-4 in cellular experiments, as shown in Figures 4.11 and 4.12. The increased brightness of Fluo-8 is partly attributable to its superior loading in most cell types. Although a lower pKa was assumed necessary when the Fluo dyes were invented, experimental data has established that Fluo-8’s pKa is not a limitation.
TEFLabs Fluo-8 (and thus Fluo-2 Medium Affinity) offer improved cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelength of maximum excitation at 490 nm and maximum emission at 520 nm. Fluo-8 loadng can be performed at room temperature while Fluo-3 AM and Fluo-4 AM may require 37 degree C cell loading.
TEFLabs offers Low Affinity, High Affinity, Leakage Resistant, and Near Membrane versions of Fluo-8 under the original Fluo-2 name.
For improved performance over Fluo indicators, TEFLabs recommends Asante Calcium Green (substantially enhanced brightness and dynamic range) and, for some applications, Asante Calcium Red (fluorescence emission at much longer wavelength).
DATA
Excitation: 488 nm
Emission: 515 nm
kd: 390 nM
Solubility: H20 (salt form) DMSO (AM form)
Molecular Weight: 877 g/mol (salt) 1047 g/mol (AM)
RESULTS
The protocol for using Fluo-2 (TEFLabs Fluo-8) is the same that is used for Fluo-4.Typical loading protocol for Fluo-2 (TEFLabs Fluo-8)
- Prepare stock solution of 1 – 10 mM Fluo-8 (AM) in DMSO
- Dilute AM stock solution to 1 – 5 μM in aqueous medium and incubate cells for 30 – 60 minutes at room temperature. (If necessary, premix Pluronic F-127 stock solution (15% wt/vol in DMSO) with the desired volume of Fluo-2 (AM) stock solution before dispersal into aqueous medium.)
- Wash/transfer cells into fresh medium for experimentation (for “wash-free” assays, washing may be omitted).
Figure. 4.10 Fluo-8(Fluo-2 MedAff), 3, 4 structures
Table 4.6 TEFLabs Fluo-8(Fluo-2 MedAff) versus Fluo-4 comparison
Table 4.6 shows a comparison of Fluo-2 MedAff (TEFlabs Fluo-8) and Fluo-4 properties. the pKa of Fluo-2 MedAff is slightly higher than Fluo-4. Although a lower pKa was preferred when the Fluo dyes were invented, experimental data has established that Fluo-2’s pKa is not a limitation. (See History of Fluo Dyes)
Figure. 4.11 FLUO-2,3, 4 brightness comparison
Figure. 4.12 FLUO-2, 4 cell comparison
Table 4.7 Fluo-2 MedAff/Fluo-8 properties
RELATED PRODUCTS
Fluo-2 HighAff
Fluo-2 LowAff
Fluo-2 LeakRes
Fluo-2 NearMem
Asante Calcium Red (ACR)
Asante Calcium Green
Asante NaTRIUM Green-1
Asante Potassium Green-1