T cell calcium mobilization study using Fluo-3 AM (flow cytometry)

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-CD3 Armernian 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)
  • Pluronic F-127
  • Hanks buffered solution (HBSS)
  • Dye loading buffer (see Recipes)


  • Flow cytometer


  1. Prepare dye loading buffer 2 ml for one sample.
  2. Suspend cells at 5 x 106 cells/ml in 1 ml dye loading buffer and incubate 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 protect from light.
  7. 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.


  1. HBSS/Ca/MG/FBS (Hanks buffered solution (HBSS) supplemented with 1.3 mM CaCl2 and 1.1 mM MgCl2)

    HBSS20 ml
    1 mM CaCl226 µl
    1 mM MgCl222 µl
    FBS200 µl
  2. Dye loading buffer (2 ml for one sample)


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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