Materials and Reagents

Jurkat T cells (ATCC, catalog number: TIB-152 )
DMSO (Sigma-Aldrich, catalog number: 472301 )
RPMI media 1640 (Life Technologies, Gibco^®, catalog number: 21875-034 )
Fetal Bovine Serum (FBS) (Life Technologies, Gibco^®, catalog number: 16000044 )
Penicillin/streptomycin (pen/strep) (Life Technologies, Gibco^®, catalog number: 15140-122 )
PMA (Sigma-Aldrich, catalog number: P1585 ) (Store at -20 °C)
Ionomycin (Sigma-Aldrich, catalog number: I0623 ) (Store at -20 °C)
Calcium Assay Kit (BD Biosciences, catalog number: 640176 )
Complete RPMI media (see Recipes)
PMA stock solution (see Recipes)
Ionomycin stock solution (see Recipes)

Equipment

Centrifuge (Eppendorf, model: 5810R )
37 °C 5% CO₂ Cell culture incubator
Cell Counter
BD CantoII FACS machine
5 ml round-bottom FACS tube (BD Biosciences, catalog number: 352003 )

Software

FACS DIVA software
FlowJo software

Procedure

Preparation (For 4 samples)
1. Preparation of Jurkat T cells:
  1. Count cells growing exponentially (1 x 10⁶ per assay).
  2. Wash cells once with pre-warmed complete RPMI media at 200 x g for 5 min.
  3. Place 1 x 10⁶ cells/250 μl of fresh complete RPMI media into a 5 ml FACS tube.
2. Preparation of 1x enhancing solution (from Calcium Assay Kit) (2 ml for 4 assays):
  1. Mix 200 μl of 10x enhancing solution with 1.8 ml of assay buffer.
  2. Keep it at room temperature.
3. Preparation of indicator (from Calcium Assay Kit):
  1. Equilibrate a vial of indicator (stored at -20 °C) at room temperature for 5 min.
  2. Add 100 μl of 100% DMSO.
  3. Mix well by pipetting up and down multiple times.
  4. Store at RT for 10 min to stabilize completely.
  5. Store unused indicator in a small aliquot at -20 °C until use.
4. Preparation of 1x loading dye:
  Mix 2 ml of 1x enhancing solution with 2 μl of indicator prepared above.
5. Loading dye to cells:
  Add 250 μl of 1x loading dye (prepared in step 4) into each tube containing cells.
6. Incubate tube for 1 h in 37 °C CO₂ Incubator.
7. Cool down tube at RT for 20 min before analysis
FACS analysis
1. Open the FACS DIVA software.
2. Draw a dot plot (Time is on the X-axis, and FITC is on the Y-axis).
3. Place a tube to the FACS machine.
4. Click “Record Data” for 1 min to obtain the basal level of signal.
5. Click “Stop Acquiring” and remove the tube from the FACS machine.
6. Immediately add 1 μl of stimulator [a mixture of PMA (50 ng/ml) and ionomycin (1 μg/ml)].
7. Vortex briefly and place the tube back to the FACS machine.
8. Click “Record Data” for additional 3 min.
9. Click “Append” to attach acquired signal to the 1 min basal level of signal acquired in step 4.
10. Click “Stop Acquiring” to finish the assay.
11. Analyze each data with FlowJo software by choosing kinetic mode.
12. Overlay sample data on the control data to display the difference of signal between the control and sample on one Figure as shown in Figure 1.
  
  Figure 1. Intracellular Ca²⁺ influx in IKKγ/NEMO-deficient Jurkat T cells stably complemented with Ha-tagged IKKγ/NEMO WT or mutants (S377E, S377A, Y374D, Y374F). Cells were loaded with Ca²⁺ indicator for 1 h at 37 °C. Intracellular Ca²⁺ influx upon PMA and ionomycin (P + I) treatment was monitored for 5 min by flow cytometry analysis. Vec stands for vector control.

Recipes

Complete RPMI media
RPMI containing 10% FBS and 1% penicillin/streptomycin
PMA stock solution
50 ng/ml DMSO
Ionomycin stock solution
1 μg/ml DMSO

Acknowledgments

This protocol is adapted from Lee et al. (2012).

References

Lee, S. H., Toth, Z., Wong, L. Y., Brulois, K., Nguyen, J., Lee, J. Y., Zandi, E. and Jung, J. U. (2012). Novel Phosphorylations of IKKγ/NEMO. MBio 3(6): e00411-00412.