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. Author manuscript; available in PMC: 2016 Nov 1.
Published in final edited form as: Biochim Biophys Acta. 2015 Aug 8;1850(11):2318–2328. doi: 10.1016/j.bbagen.2015.08.002

Fig. 2. SypHer-2 detects activity-associated pH oscillations in a dissociated primary neuronal culture.

Fig. 2

A) Time lapse imaging of a cultured mouse hippocampal neuron using SypHer-2 (upper row) for pH and R-GECO (lower row) for calcium measurements. Numbers indicate time in seconds, lookup table reflects SypHer-2 ratio. Scale bar is 40 μm; B), C) Intracellular pH and calcium levels were registered in neuronal cell bodies using SypHer-2 (black) and R-GECO (red) respectively. pH oscillations were associated with calcium entry into cells, and both were inhibited in the presence of 1mM BAPTA-AM (B) or 100 nM TTX (C); D) After each experiment SypHer-2 ratiometric signal was calibrated using ionophore containing buffers. Numbers indicate pH values; E) Dynamics of intracellular synthetic indicator BCECF-AM fluorescence, excited by 504 nm light, confirms existence of pH oscillations. All experiments were replicated with 3 to 5 neuronal cells from two different preparations.