T Cell Calcium Mobilization Study (Flow Cytometry)    

Materials and Reagents

1. Anti-CD3 Armernian hamster primary antibody (BD Biosciences, catalog number: 553057 , NA/LE)
   
2. Goat anti-Armernian Hamster IgG antibody (Jackson ImmunoResearch, catalog number: 127-005-099 )
   
3. Phosphate buffered saline (PBS)
   
4. CaCl₂
   
5. MgCl₂
   
6. DMSO (Merck KGaA, Calbiochem^®, catalog number: 540025 )
   
7. Fluo-3 AM in DMSO (Life Technologies, Molecular Probes^®, catalog number: F-1242 )
   (Note: Fluo-3 fluorescence is calcium-dependent) (see Recipes)
   
8. Fura Red AM in DMSO (Life Technologies, Molecular Probes^®, catalog number: F-3020 ) (Note: Fura Red fluorescence is calcium-independent. Fura Red serves as a control of dye loading efficiency) (see Recipes)
   
9. Pluronic F-127 in DMSO (Life Technologies, Molecular Probes^®, catalog number: F-1242) (see Recipes)
   
10. Hanks buffered solution (HBSS) (Life Technologies, Gibco^®, catalog number: 14170-112 ) (see Recipes)
    
11. Dye loading buffer (see Recipes)
    

Equipment

1. Flow Cytometry

Procedure

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.
   

Recipes

1. Dye loading buffer (2 ml for one sample)
   1. HBSS/Ca/Mg/FBS

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

   2. Dye loading buffer

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

2. Hanks buffered solution (HBSS) supplemented with 1.3 mM CaCl₂ and 1.1 mM MgCl₂.
   
3. 10% (w/v) pluronic F-127 in DMSO, 10%, store at RT.
   
4. 4 mg/ml Fluo-3 AM in DMSO (20 x 50 μg) aliquot and store in a -20 °C dessicator.
   
5. 10 mg/ml Fura Red AM in DMSO (500 μg) aliquot and store in a -20 °C dessicator.
   

Acknowledgments

This protocol was previously used in and adapted from Huang et al. (2008).

References

1. Huang, G. N., Huso, D. L., Bouyain, S., Tu, J., McCorkell, K. A., May, M. J., Zhu, Y., Lutz, M., Collins, S., Dehoff, M., Kang, S., Whartenby, K., Powell, J., Leahy, D. and Worley, P. F. (2008). NFAT binding and regulation of T cell activation by the cytoplasmic scaffolding Homer proteins. Science 319(5862): 476-481.