Fluo 3 is a long wavelength calcium probe that is practically nonfluorescent in its free ligand form, but its fluorescence increases 60-80 times when it forms complexes with calcium. Thus, it has been widely used with confocal laser fluorescent microscopy because the microscope has an argon laser. The long wavelength of the fluorescent signal is also convenient for minimizing photodamage to sample cells. Fluo 3 is also useful for caged calcium and others that are cleaved by the photoirradiation in the UV region. Fluo 3-AM is an acetoxymethyl ester derivative of Fluo 3 that can be easily loaded into cells by incubation.
General Protocol (for Human T cells)*
・2 mM Fluo 3-AM/DMSO (1 mg Fluo 3-AM in 442 μl DMSO)
・Hanks Ebalanced salt solution (HBSS)
・HEPES buffer saline (10 mM HEPES, 1 mM Na2HPO4, 137 mM NaCl, 5 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, 0.1% BSA, pH 7.4)
1. Add 16.5 mg Pluronic F127 to Fluo 3-AM/DMSO solution. Pluronic F127 prevents aggregation of Fluo 3-AM in HBSS and helps uptake with cells.
2. Dilute the Fluo 3-AM solution with HBSS to prepare 4 μM Fluo 3-AM working solution.
3. Add the Fluo 3-AM working solution to the cells and incubate at 37oC for 20 min.
4. Add HBSS containing 1% fetal Calf serum equivalent to 5 times the volume of Fluo 3 -AM working solution (step 3).
5. Wash the cells 3 times with HEPES buffer saline. Then resuspend the cells to prepare 1×105 cells per ml solution using HEPES buffer saline.
6. Incubate at 37oC for 10 min. Then use the cells for fluorescent calcium ion detection.
7. Monitor the fluorescence at 528 nm (excitation: 490-500 nm).
*Cell staining conditions depend on cell types, so it is necessary to optimize the conditions for each experiment.
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