Calcium imaging and analysis

Optical recordings of intracellular [Ca²⁺] indicated by jRGECO1a fluorescence were performed by placing cultures in a mini incubator (Bioscience Tools) kept at 37 °C with a constant supply of humidified blood gas (5% CO₂, 21% oxygen, balanced nitrogen, airgas) on a stage of a fluorescent inverted microscope (Olympus). Changes in fluorescence were observed via ×4 objective at 60 ms exposure and camera frame rate of 200 ms/frame. Optically recorded data were analyzed using ImageJ and MATLAB.

ROI were drawn either around the whole visible culture or portions of interest. Mean fluorescence in that ROI was calculated for each time point. The baseline was then determined using algorithm developed by Eilers and Boelens, 2005 for each time point. Mean fluorescence was then converted to ΔF/F. If raw gray value data is R and baseline is F₀, then:

Activity was then detected by thresholding (3 × std) ΔF/F values. Cultures were recorded on DIV 10, 12, 14, and 16.