Dissociation and Culture of Rat DRG Neurons

DRGs from adult male Sprague-Dawley rats were dissected and dissociated as described previously (Calcutt et al., 2017 (link); Sabbir et al., 2018 (link)) and cultured in defined Hams F12 media containing 10mM D-glucose (N4888, Sigma, St Louis, MO, USA) supplemented with modified Bottenstein's N2 additives without insulin (0.1 mg/ml TF, 20 nM progesterone, 100 μM putrescine, 30 nM sodium selenite, 0.1 mg/ml BSA; all additives were from Sigma, St Louis, MO, USA) (Akude et al., 2011 (link); Roy Chowdhury et al., 2012 (link); Saleh et al., 2013 (link); Calcutt et al., 2017 (link)). In all experiments, the media was also supplemented with 0.146 g/L L-glutamine, a low dose cocktail of neurotrophic factors (0.1 ng/ml NGF, 1.0 ng/ml GDNF and 1 ng/ml NT-3 – all from Promega, Madison, WI, USA), 0.1 nM insulin and 1X antibiotic antimycotic solution (A5955, Sigma).