The following protocol uses non-ratiometric dye Fluo-3 AM, combining it with flow cytometry to monitor what modifications the T cell receptor-triggered calcium exhibits over time.
Materials and Reagents
- Anti-CD3 Armernian hamster primary antibody
- Goat anti-Armernian Hamster IgG antibody
- Fluo-3 AM (Note: Fluo-3 fluorescence is calcium-dependent) (See recipes section)
- Fura Red AM (Note: Fura Red fluorescence is calcium-independent. Fura Red serves as a control of dye loading efficiency)
- Hanks buffered solution (HBSS)
- Pluronic F-127
- Dye loading buffer (see Recipes)
- For one sample, prepare dye loading buffer 2 ml
- Cells have to be suspended at 5 x 106 cells/ml in 1 ml dye loading buffer, then incubated for 30 min at 37 °C
- Spin down cells 5 min at 1,000 rpm
- Stain cells with 5 μg/ml anti-CD3 hamster primary antibody (no azide) for 30 min on ice or at 4 °C (0.5 μg/100 μl PBS)
- Wash cells once
- Resuspend cells in 3 ml HBSS/Ca/Mg/FBS at 3 x 106 cells/ml and store at RT and keep away from light
- In order to achieve calcium mobilization, samples have to be warmed up at 37 °C for 5-10 min. Then, flow cytometry needs to be applied to the samples to measure the calcium baseline. Five minutes later, add 5 μg/ml goat anti-hamster IgG antibody (15 μg/3 ml), mix well and immediately continue the measurement with flow cytometry. To maintain the incubation temperature, a small beaker containing water that has been warmed in advance to 37 °C is needed to bath sample tubes while they are being measured.
1. HBSS/Ca/MG/FBS (Hanks buffered solution (HBSS) supplemented with 1.3 mM CaCl2 and 1.1 mM MgCl2)
|1 mM CaCl2
|1 mM MgCl2
2. Dye loading buffer (2 ml for one sample)
|10% pluronic F-127 (DMSO)
|4 mg/ml Fluo-3 AM (DMSO)
|10 mg/ml Fura Red AM (DMSO)
This protocol is adapted from Huang, G. N. (2012). T cell Calcium Mobilization Study (Flow Cytometry). Bio-protocol 2(9): e171. http://www.bio-protocol.org/e171
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