2.12. Calcium imaging of mammalian sensory neurons

Calcium imaging of mouse dorsal root ganglion (DRG) sensory cells was performed as previously described [24]. In brief, DRG cells isolated from 4 to 8-week-old male CS7BL/6 mice (under The University of Queensland Animal Ethics Committee approval TRI/IMB/093/17) were dissociated and plated in Dulbecco’s modified Eagle’s medium (ThermoFisher Scientific, Grand Island, NY, USA) containing 10% FBS (Assaymatrix, Melbourne, Australia) and penicillin/streptomycin (ThermoFisher) on a 96-well poly-D-lysine--coated culture plate (Corning), and maintained overnight. Cells were loaded with Fluo-4 AM calcium indicator as per the manufacturer’s instructions (ThermoFisher). After loading for 1 h, the dye solution was replaced with assay solution (Hanks’ balanced salt solution and 20 mM HEPES). Fluorescence corresponding to intracellular calcium ([Ca²⁺]_i) of typically 100–150 DRG cells per experiment was monitored in parallel using a Nikon Ti-E deconvolution inverted microscope, equipped with a Lumencor Spectra LED light source. Images were acquired using a 20 × objective at one frame/s (excitation 485 nm; emission 521 nm). For each experiment, baseline fluorescence was monitored for 30 s and then a wash of assay solution was applied. At 60 s, the assay solution was replaced with assay solution containing individual peptides (10 μM) and the cells were observed for a further 90 s.