Primary DRG Neuron Calcium Imaging

Primary cultures of DRG neurons were generated from C57BL/6N mice as described previously (65 (link)). Briefly, DRG was incubated in collagenase/dispase (5 mg/ml) (10269638001, Millipore-Sigma) for 30 min, and cells were mechanically dissociated, seeded on poly-d lysine–coated coverslips, and cultured for 48 hours [Dulbecco’s modified Eagle medium (DMEM)/F12, 10% fetal bovine serum (FBS), penicillin/streptomycin, nerve growth factor (100 ng/ml), and glial cell line–derived neurotrophic factor (50 ng/ml)]. For calcium imaging, DRG neurons were transfected with AAV9.Syn.GCaMP6f.WPRE.SV40 (2 μl, 1 × 10¹³ μg/ml, AV-9-PV2824) (66 (link)). After 48 hours of culture, coverslips with DRG neurons expressing the calcium indicator GCaMP6f were mounted on a DMi8 microscope (Leica) in HBSS buffer (140 mM NaCl, 2 mM CaCl₂, 10 mM Hepes, 4 mM KCl, and 1 mM MgCl₂ (pH 7.4)] and constantly superfused from a gravity-fed six-channel system (VC-6, Warner Instruments). Imaging was performed with an ORCA-Flash 4.0 C1440 digital complementary metal-oxide semiconductor camera (Hamamatsu, Bridgewater, NJ) at 1 Hz. Fluorescence intensity in hand-drawn regions of interest was extracted using HCImage (Hamamatsu) and plotted against time. The R package Pheatmap was used to generate a heatmap plot to visualize fluorescence signals in all the neurons.