Fluo-3 AM (Flow Cytometry) for T Cell Calcium Mobilization: A Study

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)


Flow cytometer


  1. For one sample, prepare dye loading buffer 2 ml
  2. 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
  3. Spin down cells 5 min at 1,000 rpm
  4. 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)
  5. Wash cells once
  6. Resuspend cells in 3 ml HBSS/Ca/Mg/FBS at 3 x 106 cells/ml and store at RT and keep away from light
  7. 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)

. .
HBSS 20 ml
1 mM CaCl2 26 µl
1 mM MgCl2 22 µl
FBS 200 µl


2. Dye loading buffer (2 ml for one sample)

. .
HBSS/Ca/Mg/FBS 2 ml
10% pluronic F-127 (DMSO) 4 µl
4 mg/ml Fluo-3 AM (DMSO) 2 µl
10 mg/ml Fura Red AM (DMSO) 2 µl



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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Last updated: May 21, 2019 at 6:45 AM


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