Extracellular Ca²⁺ plays a crucial role in the cl-CD95L-mediated Ca²⁺ rise.

(A) Indicated T-cells were loaded with 1 μM of the calcium probe Fura-2AM for 30 min at RT. Cells were bathed at 37°C in a medium containing 2 mM Ca2+ (2 mM [Ca2+]e) or a Ca2+-free medium (0 mM [Ca2+]e) and then treated with supernatant of pcDNA3.1(+)-transfected HEK cells (Untreated) or with 100 ng/ml of cl-CD95L. Values were recorded every 10 s. Regions of Interest (ROIs) outlining individual cells were defined in three independently performed experiments and the mean ratio in each ROI was plotted versus time. Ratio values were converted to [Ca2+]i values using a calibration curve (see Materials and Methods section). For each treatment, the mean ± SD of the area under the curve (AUC) measured for 1,000 s in individual cells is depicted. Basal and maximal levels of [Ca2+]i are indicated by red and blue dotted lines, respectively. (B) Statistical analyses of the AUC values for indicated cells incubated in the presence of cl-CD95L in a regular (2 mM) or a Ca2+-free medium (0 mM). Using non-parametric two-tailed Mann-Whitney test, *** indicates a p value ≤0.001. (C) PBLs were untreated (DMSO, upper panel) or treated (lower panel) with the SOC channel inhibitor BTP-2 (500 nM) and then 100 ng/ml of cl-CD95L was added. For each condition, the mean ± SD of the area under the curve (AUC) measured for 1,000 s is depicted. Basal and maximal levels of [Ca2+]i are indicated by red and blue dotted lines, respectively. Note that individual calcium oscillations and/or synchronous calcium oscillations (upper panel) lead to apparent calcium oscillations during the plateau phase. (TIF)