Calcium Flux Analysis of Human T-Cells

Calcium flux analyses of human T-cells were performed according to^{67 (link)}. Briefly, 3 × 10⁵ cells/mL in phenol red-free RPMI 1640 (Life Technologies) containing 10% FCS were loaded with 5 µg/mL Indo-1 AM (MoBiTec, Göttingen, Germany) at 37 °C for 45 min, followed by an additional incubation for 45 min in medium without Indo-1. Cells were kept on ice before equilibration at 37 °C for 5 min, directly before measurement. Changes in intracellular calcium were monitored using a flow cytometer LSRI (BD Biosciences, Heidelberg, Germany). Cells were illuminated using the 325 nm laser-line of a helium-cadmium laser. Fluorescence emissions at 405/30 nm (calcium-bound Indo) and 510/20 nm (free Indo) were detected simultaneously, analyzing the ratio of bound to free Indo over time. After monitoring the baseline activity for 1 min, the cells were stimulated by 10 µg/mL CD3 mAb (clone UCHT1, BD Biosciences) and cells were measured for another 6 min. To confirm proper Indo-1 loading, the cells were then treated with 10 µg/mL ionomycin (Sigma-Aldrich). Kinetics were analyzed using FlowJo v7.6.3 software (Tree Star, Ashland, USA).