Fig. 3. Extracellular [Ca²⁺] measured with a near-membrane fluorescent indicator in CaR-expressing HEK 293 cell clusters. The figure shows results of experiments in which cells were initially bathed in a low [Ca²⁺] solution containing a low concentration of the high-affinity Ca²⁺ chelator BAPTA (25 µM; a). This allowed detection of extracellular [Ca²⁺] changes by a high-affinity near-membrane fluorescent Ca²⁺ indicator, fura-C₁₈. Stimulation of intracellular Ca²⁺ signaling through CaR using 1 mM spermine (b) results in an increase in the fura-C₁₈ ratio (red pseudocolor) in the diffusion-limited clefts between cells, which is indicative of an increase in extracellular [Ca²⁺]. Extracellular [Ca²⁺] is reduced even further in the presence of 1 mM BAPTA (c). (d) Membrane localization of the fluorophore (fluorescence at 340 nm excitation, emission 510 nm). For additional details, see De Luisi and Hofer (De Luisi and Hofer, 2003).