DRG from 4- to 8-week-old male C57BL/6 mice were dissociated and plated in Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum (FBS; Assaymatrix) and penicillin/streptomycin (Gibco) on a 96-well poly-d-lysine–coated culture plate (Corning) and maintained overnight. Cells were loaded with Fluo-4 AM calcium indicator, according to the manufacturer’s instructions (Thermo Fisher Scientific). After loading (1 hour), the dye-containing solution was replaced with assay solution (1× Hanks’ balanced salt solution and 20 mM Hepes). Fluorescence corresponding to [Ca2+]i of typically 100 to 150 DRG cells per experiment was monitored in parallel using a Nikon Ti-E Deconvolution inverted microscope, equipped with a Lumencor Spectra LED Lightsource. Images were acquired at a 20× objective at 1 frame/s (excitation, 485 nm; emission, 521 nm). For each experiment, baseline fluorescence was monitored for 20 s, and at 30 s, the assay solution was replaced with an assay solution containing individual peptides [1:10 dilution of resuspended fraction (described above)] or whole venom (1:1000 dilution). Experiments involving the use of mouse tissue were approved by The University of Queensland animal ethics committee.

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