Fig. 4. Measurement of extracellular near-membrane [Ca²⁺] with fura-C₁₈. (A) Images a-c depict ratio images of fura-C₁₈-loaded HEK CaR cells taken at times corresponding to points marked with arrows on the trace in panel B in four regions (indicated by black circles). Image d shows fluorescence at 340 nm excitation (510 nm emission) of the same fura-C₁₈ loaded cells. (B) Fura-C₁₈ ratio change of HEK CaR cells from regions shown in panel A in response to 1 mM spermine, followed by superfusion with 1 mM BAPTA and then 1 mM Ca²⁺. Inset shows enlarged time-scale of fura-C₁₈ ratio from a selected region in the same experiment. The intracellular [Ca²⁺] signal as measured by fura-2 from a typical trace in a separate experiment is overlaid to illustrate the differences in time course of the two responses. (C) Example of an experiment in which extracellular [Ca²⁺] oscillations were detected following stimulation with 1 mM spermine in HEK CaR cells. (D) Significantly larger extracellular transients were detected in HEK CaR cells in response to stimulation with carbachol (CCh; 100 µM) and ATP (100 µM).