Nuclear and cytoplasmic calcium signals are not affected by the mitochondrial dye. A, neurons were incubated with the mitochondria-selective dye MitoTracker Deep Red 633 (red) and the DNA specific stain HOECHST 33258 (blue) for 20 min in SGG. Scale bar is 20 μm. C and D, no apparent differences in network properties were detected in hippocampal neurons loaded with Fluo-4 and MitoTracker Deep Red 633. Periodic calcium transients were induced by adding 50 μM of the GABAA receptor blocker bicuculline (B). Neurons were depolarized by exposing the cells to 50 mM KCl (C). Images were taken every 1.6 sec. Measurements of individual cells (thin grey lines) and their mean (bold black line) are shown. D, periodic calcium transients (induced by 50 μM bicuculline) in hippocampal neurons loaded with Fluo-4 and MitoTracker were measured in the nucleus (continuous line) and cytosol (dotted line). The average of n = 7 cells is shown. Image acquisition rate was 0.6 Hz.