| BACKGROUND/TECHNICAL INFORMATION |
General Protocol (for Human T cells)
Reagents:
2mM Fluo 3/AM/DMSO (1mg Fluo 3/AM in 442μl DMSO)
Pluronic F127
Hanks-balanced salt solution (HBSS)
HEPES buffer saline (10mM HEPES, 1mM Na2HPO4, 137mM NaCl, 5mM KCl, 1mM CaCl2, 0.5mM MgCl2, 5mM glucose, 0.1% BSA, pH 7.4) Protocol:
1. Add 16.5mg 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 ºC for 20min.
4. Add 5 volumes of HBSS containing 1% fetal calf serum, and continue the incubation for another 40min.
5. Wash the cells 3 times with HEPES buffer saline. Then resuspend the cells to prepare 1x105 cells/ml solution using HEPES buffer saline.
6. Incubate at 37ºC for 10min. Then use the cells for fluorescent calcium ion detection.
7. Monitor the fluorescence at 528nm (excitation: 490-500nm) Cell staining conditions differ by cell types, so it is necessary to optimize the conditions for each experiment.
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