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CAS:273221-67-3
Appearance:Orange Solid
Storage:Powder : -20℃, 1 year;In solvent(mother liquid): -20℃, 1 month; -80℃, 6 months
Purity:≥95%
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Fluo-4 is a calcium fluorescence probe that replaces Cl in the Fluo-3 structure with F. By replacing Cl with F, which is more electrically attractive, its maximum excitation wavelength will deviate by about 10 nm towards the short wavelength. This wavelength is closer to the wavelength of an argon laser, so when excited by an argon laser, the fluorescence intensity of Fluo-4 is twice that of Fluo-3. After entering the cell membrane, Fluo-4 and AM are cleaved by the intracellular esterase to form Fluo-4, which is retained in the cell. Fluo-4 is almost non-fluorescent when it exists in the form of free ligand, but when it combines with intracellular calcium ions, it can produce strong fluorescence, and the maximum excitation wavelength is 494nm. The maximum emission wavelength is 516nm. Changes in intracellular calcium concentration can be detected using laser confocal microscopy or flow cytometry.
How to use (for reference only) :
Fluo-4 AM solution was diluted with HBSS to prepare 4-5μM of Fluo-4 AM working solution.
Fluo-4, AM working solution was added to cells and cultured at 37℃ for 20 minutes.
Five times the volume of HBSS containing 1% fetal bovine serum was added, and the culture was continued for 40 minutes.
The cells were washed with HEPES buffer saline for 3 times, and then the cells were re-suspended with HEPES buffer saline to produce 1×105 cells/mL solution.
The cells were cultured at 37℃ for 10 minutes, and then fluorescent calcium ion detection was performed. The excitation wavelength is 506nm and the emission wavelength is 526nm.
* The conditions for labeling vary depending on the cell type, so please determine the best conditions before each experiment. The above methods are for reference only.
Note:
1. If the medium containing serum is used, the lipase in the serum will decompose the AM body, thereby reducing the effect of Fluo-4 and AM entering the cell.
2, the medium containing phenol red will make the background value slightly higher, so before adding the working liquid, the medium residue should be removed as far as possible.
3. Pluronic F127 can prevent Fluo-4 and AM from polymerizing in HBSS and help it enter cells. An appropriate amount of Pluronic F127 solution can be added to Fluo-4,AM solution, but it is not recommended to store Fluo-4,AM solution in Pluronic F127 for a long time.
4. Fluorescent dyes have extraction problems, please pay attention to avoid light to slow down fluorescence extraction.
