Fig. 5. Luminal Ca²⁺ imaging in cultured hippocampal neurons loaded with mag-fura-2 AM (A) and fluo-5N AM (B) and permeabilized with ß-escin. (Top left) RFP-KDEL-labeled vesicles, (bottom left) Ca²⁺ indicators. Scale bar, 10 µm. (Top right) Time-course plots of changes in fluorescence intensity of the Ca²⁺ indicators, (right bottom) time-course plots of changes in the ratio (F₃₄₀/F₃₈₀ for mag-fura-2 and F/F₀ for fluo-5N). The ratio plots are indicated after high-cut filtering with a Gaussian filter in order to reduce the noise. The neurons were subjected to Ca²⁺ uptake (1 mM MgATP), washout of MgATP and Ca²⁺ release (20 µM Ins(1,4,5)P₃), as indicated by the horizontal boxes above the traces.