Quantifying Intracellular Calcium Dynamics

Ca²⁺ influx was assessed by real-time flow cytometry, as previously described (Martin et al., 2014 (link)). Briefly, cells were loaded with 5 mM Indo-1 AM (Molecular Probes) in the presence of 2.5 mM probenecid (PowerLoad; Molecular Probes), washed, and surface-stained for CD4 and CD8 detection. Cells were analyzed in real-time with a FACS ARIA II flow cytometer (BD Biosciences). During acquisition, 1 mg/ml of anti-CD3 antibody was added to the cells, followed by 10 μg/ml of F(ab′)2 rabbit–anti-mouse IgG crosslinker (Jackson Immunoresearch), and finally incubated with a calcium ionophore (1 mM ionomycin [Sigma-Aldrich]). Changes in the intracellular calcium concentration are quantified by a shift in the indo-1 emission peak from 485 nm (indo-blue) for unbound dye to 405 nm (indo-violet) when the indo-1 molecule is bound to calcium. Data were analyzed by using the kinetic tool of FlowJo software. Intracellular Ca²⁺ levels correspond to the normalized ratio of 405- to 485-nm indo-1 emission peaks.