T Cell Activation and Calcium Flux Assay

After transfection for 40 h, primary CD3⁺ T cells were stimulated using Dynabeads™ Human T Activator CD3/CD28 (bead to cell ratio, 1:4; Thermo Fisher). Proliferation and apoptosis were carried out according to previously described methods (49 (link)). In order to detect calcium (Ca²⁺) flux, CD3⁺ T cells were stained with LIVE/DEAD™ dye (Thermo Fisher), followed by loading with Fluo-4-AM, Fura-red, and Pluronic F-127 (Thermo Fisher) for 30 min. Cells were then labeled with antibodies against CD3, CD4, and CD8 for 20 min. To record Ca²⁺ flux, a baseline fluorescence signal was acquired for 90 s. Following this period, the cells were stimulated with soluble anti-CD3/CD28 (10 μg/mL) (50 (link)), after which fluorescence signal recording was continued for 300 s. Ionomycin (1 μg/mL) was used to elicit a maximum response for another 120 s. For quantification of Ca²⁺ flux, the ratio of Fluo-4 or Fura-red at the peak of the response relative to the baseline was determined (51 (link)). Cells were detected using the LSR II flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo software (Ashland, OR, USA).