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. Author manuscript; available in PMC: 2018 May 1.
Published in final edited form as: J Neurochem. 2017 Dec 27;145(3):188–203. doi: 10.1111/jnc.14255

Figure 4. Generation of stable inducible neurons expressing GCaMP6f for calcium imaging.

Figure 4

(a) Schematic of the pMinitol2-derived vectors that allow for generation of stable clones with the GCaMP6f under a CAG promoter, which allows for monitoring calcium status of cells. (b) Time-course of differentiation showing the generation of siNeurons that includes plating at day-1, dox treatment in E8 media for 2 days, media change to 3N with dox for additional 4 days, and finally removal of dox for analysis. (c) Brightfield, MAP2 (red), DAPI (blue) and MAP2/DAPI composite images of siNeurons differentiated with dox for 11 days. (d) Tuj1 (green), and EdU (red) staining after EdU labelling for 2.5h. (e) Quantification of number of cells incorporating EdU in the time course showing 98.8% cells are post-mitotic at day 6. The data is representative of three independent experiments (n=3) (f) Quantitative mRNA expression showing high inductions of NEUROG2, SYN and vGLUT1 upon induction with dox as normalized to GAPDH internal control for two independent cell lines. Error bars represent the standard deviation between the triplicate samples and are representative of three independent experiments.