This protocol uses non-ratiometric dye Fluo-3 AM, in combination with flow cytometry, to monitor T cell receptor-triggered calcium changes over time.
Materials and reagents
Anti-CD3Armernian hamster primary antibody
Goat anti-Armernian Hamster IgG antibody
Fluo-3 AM (Note: Fluo-3 fluorescence is calcium-dependent) (see Recipes)
Fura Red AM (Note: Fura Red fluorescence is calcium-independent. Fura Red serves as a control of dye loading efficiency)
Hanks buffered solution (HBSS)
Dye loading buffer (see Recipes)
Prepare dye loading buffer 2 ml for one sample.
Suspend cells at 5 x 106 cells/ml in 1 ml dye loading buffer and incubate 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 protect from light.
For calcium mobilization, warm up samples at 37 °C for 5-10 min. Submit samples to flow cytometry for calcium baseline measurement. After 5 min, 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 prewarmed to 37 °C is necessary to bath sample tubes during the time course of measurement.
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
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