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Fluorescence Technology

Fluorescent Calcium Indicators

Fura-2

Fura-2 is one of the most widely used calcium indicators for ratiometric measurement. Many types of instrumentation are now available for experiments using Fura-2. Fura-2 is especially suitable for digital imaging microscopy. It is less susceptible to photobleaching than Indo-1. Changes in the cell shape can sometimes affect the fluorescent ratio at 340 nm and 380 nm. For example, fluorescent signal intensities at these wavelengths sometimes decrease simultaneously with smooth muscle contraction. For blood vessels, however, the increase of the signal intensity at 340 nm tends to be smaller on contraction, while the decrease of the signal intensity at 380 nm tends to be larger with its contraction. Fura-2-AM is an acetoxymethyl ester derivative of Fura-2 that can be easily loaded into cells by incubation.

General Protocol (for NG 108-15/ Neuronal Cell Line)

Reagents
1 mM Fura-2-AM/DMSO (1 mg Fura 2-AM in 1 ml DMSO)
Hanks’ balanced salt solution (HBSS)
HEPES buffer saline (20 mM HEPES, 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgCl₂, 13.8 mM glucose, pH 7.4)

Protocol

1. Culture cells on a glass-bottom dish using DMEM containing 5% fetal calf serum.
   
2. Change the medium to 1 mM dibutyl cAMP/DMEM, and culture the cells for 3-4 days to induce dendrites.
   
3. Dilute 1 mM Fura-2-AM DMSO solution with HEPES buffer saline to prepare 1 mM Fura-2-AM working solution.
   
4. Remove the culture medium, and add 0.5 ml of the Fura-2-AM working solution to the cells.
   
5. Incubate for 20 min. Then remove the Fura 2-AM working solution.
   
6. Wash the cells once with HEPES buffer saline. Then incubate the cells for 1 hour in the HEPES buffer saline.
   
7. Use the cells for fluorescent calcium ion detection.
   
8. Monitor the excitation spectra at 380 nm (calcium free) and 340 nm (calcium complex) with fixed emission at 510 nm.
   

*Cell staining conditions differ by cell types, so it is necessary to optimize the conditions for each experiment.

References
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Fluo Indicators

Fluo-3 is a long wavelength calcium probe. Fluo-3 is practically non-fluorescent 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 photo-irradiation in the UV region. Fluo-3-AM is an acetoxymethyl ester derivative of Fluo-3 that can be easily loaded into cells by incubation.



Fig. 2: Calcium Chelation

General Protocol (for Human T cells)*

2 mM Fluo-3-AM/DMSO (1 mg Fluo-3-AM in 442 ml DMSO)

Pluronic F127

Hanks’ balanced 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)

Protocol

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 mM Fluo 3-AM working solution.
   
3. Add the Fluo 3-AM working solution to the cells, and incubate at 37°C for 20 min.
   
4. Add 5 volumes of HBSS containing 1% fetal calf serum, and continue the incubation for another 40 min.
   
5. Wash the cells 3 times with HEPES buffer saline. Then resuspend the cells to prepare 1 x 10⁵ cells/ml solution using HEPES buffer saline.
   
6. Incubate at 37°C 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 differ by cell types, so it is necessary to optimize the conditions for each experiment.

References
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