ATP-induced intracellular Ca²⁺ increase is mediated by both P2X and P2Y receptors’ activation in DC2 cells. (A) Schematic representation of the calcium fluorometric assay in DC2 cells using the microfluidic BioFlux imaging system (left panel). Real-time ATP-induced Ca²⁺_i response was measured over time using the fluorescent calcium-sensitive dye, Cal-520 AM (5 µm). Images in the right panels show Cal-520 fluorescence before (baseline) and after addition of ATPγS. (B) Stimulation with 100 µm ATPγS, a nonhydrolyzable form of ATP, triggered a rapid and transient Ca²⁺_i increase (ATPγS). This ATP-elicited calcium response was greatly reduced in the absence of external calcium (Ca²⁺-free solution + EGTA 1 m m; ATPγS Ca²⁺-free). (C) Comparison of the maximal Ca²⁺_i response in the presence or absence of external Ca²⁺ revealed a significant decrease under Ca²⁺-free conditions. Intracellular Ca²⁺ level was normalized for baseline (corresponding to the values before the addition of ATPγS). Data are shown as mean ± SEM (n = 4). ^∗P = .0286 by the Mann-Whitney nonparametric test.