Isolation and Culture of DRG Neurons

Techniques for isolation and DRG cell culturing were similar to those previously described (Dembla et al., 2017 (link)). Briefly, DRGs were harvested from all segments and placed into chilled HBSS (Thermo Fisher Scientific, Karlsruhe, Germany). Isolated ganglia were partially digested for 40 min in 1.8 U/ml liberase DH Research Grade (Roche, Mannheim, Germany) at 37°C and digestion was stopped by adding 1 ml culture medium consisting of DMEM, 10% FBS and 1x penicillin-streptomycin (all from Thermo Fisher Scientific, Karlsruhe, Germany). DRGs were triturated with a 1,000 μl pipette and washed once with culture medium. After centrifugation, the supernatant was discarded, and the cells were suspended in culture medium. Subsequently, one tenth to one twelfth of the cell suspension was plated onto the center of a 35 mm plastic dish pre-coated with laminin (Sigma-Aldrich, Munich, Germany). The cells were left to adhere at 37°C in an incubator in a humidified atmosphere containing 5% CO₂ for 1 h after which 2 ml of culture medium were added. Cells were used for measurements on the following day.