Real-time Flow Cytometric Analysis of Ca2+ Influx

Ca²⁺ influx was assessed by real-time flow cytometry, as previously described (Martin et al., 2014 (link)). Briefly, cells were loaded with 5 µM Indo-1 AM (Molecular Probes) in presence of 2.5 mM of 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 µg ml⁻¹ anti-CD3 antibody was added to the cells, followed by 10 µg ml⁻¹ of F(ab′)₂ rabbit–anti-mouse IgG cross-linker (Jackson ImmunoResearch) and finally incubated with a calcium ionophore (1 mM ionomycin; Sigma). 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 using kinetic tool of FlowJo software (TreeStar). Intracellular Ca²⁺ levels correspond to the normalized ratio of 405 nm/485 nm indo-1 emission peaks.