Calcium imaging using Fura-2 AM and Fluo-4 AM

Fura-2 AM (Abcam, ab120873) stock solution was prepared in DMSO at 10 mM. Resting cells were loaded with the Fura-2 AM dye simultaneously with the depolarized cells at a final concentration of 2 µM. The dye was added along with the KCl solution to the depolarized cells, and the cells were incubated for 45 min at 37 °C and 5% CO₂. Then, cells were washed with neurobasal media three times and were kept in neurobasal media without Fura-2 AM at 37 °C and 5% CO₂ for another 30 min. The fluorescence was then imaged using a Keyence microscope at the excitation wavelength 340/380. Fluorescent nuclei were counted using ImageJ software.
Fluo-4 AM (Thermo Fisher Scientific, F14201) was resuspended in DMSO to 1 mM. Similar to the Fura-2 AM strategy, resting and depolarizing cells were loaded with the Fluo-4 AM at 2 µM simultaneously at the beginning of depolarization for 45 min at 37 °C and 5% CO₂. Pluronic F-127 (Thermo Fisher Scientific, P6866) was added at 0.02% to help disperse the dye in the media. Cells were then washed with regular neurobasal media three times and were kept in neurobasal media for another 30 min at 37 °C and 5% CO₂. The fluorescence was then imaged using the Keyence microscope at the excitation wavelength 494/506. Fluorescent nuclei were counted using ImageJ.
In addition to imaging, fluorescence by Fura-2 AM or Fluo-4 AM was measured using the Varioskan LUX plate reader and the SkanIt RE 5.0 program at excitation/emission at 340/380 nm and 494/506 nm, respectively. Each biological replicate was calculated as the average of three wells (technical replicates) in the 96-well plate plated from the same batch of neurons.