Neural basal media a with b27 supplement

DRGs from mice or rats were rapidly harvested following deep isoflurane anesthesia and decapitation. Ganglia were placed in a 35 mm dish containing Ca²⁺/Mg²⁺-free, cold HBBS (Life Technologies) and cut into four to six pieces that were incubated in in 0.01% blendzyme 2 (Roche Diagnostics, Indianapolis, IN) for 26 min followed by incubation in 0.25% trypsin (Sigma Aldrich, St. Louis, MO) and 0.125% DNAse (Sigma) for 30 min, both dissolved in Dulbecco’s modified Eagle’s medium (DMEM)/F12 with glutaMAX (Invitrogen, Carlsbad, CA). After exposure to 0.1% trypsin inhibitor and centrifugation, the pellet was gently triturated in culture medium containing Neural Basal Media A with B27 supplement (Invitrogen), 0.5 mM glutamine, 10 ng/ml nerve growth factor 7S (Alomone Labs, Jerusalem, Israel) and 0.02 mg/ml gentamicin (Invitrogen). Dissociated neurons were plated onto poly-L-lysine coated glass cover slips (DeutschesSpiegelglas, Carolina Biological Supply, Burlington, NC) and maintained at 37°C in humidified 95% air and 5% CO₂ for 2 hours, and were studied no later than 8 hours after harvest.

After DRGs harvesting, ganglia were placed in a 35 mm dish containing Ca²⁺/Mg²⁺-free, cold HBBS (Thermo Fisher Scientific, Waltham, MA ) and cut into four to six pieces that were incubated in 0.01% blendzyme 2 (Roche Diagnostics, Indianapolis, IN) for 26 min followed by incubation in 0.25% trypsin (Sigma Aldrich, St. Louis, MO) and 0.125% DNAse (Sigma) for 30 min, both dissolved in Dulbecco’s modified Eagle’s medium (DMEM)/F12 with glutaMAX (Invitrogen, Carlsbad, CA). After exposure to 0.1% trypsin inhibitor and centrifugation, the pellet was gently triturated in culture medium containing Neural Basal Media A with B27 supplement (Invitrogen), 0.5 mM glutamine, 10 ng/ml nerve growth factor 7S (Alomone Labs, Jerusalem, Israel) and 0.02 mg/ml gentamicin (Invitrogen). Dissociated neurons were plated onto poly-L-lysine coated glass cover slips (Deutsches Spiegelglas, Carolina Biological Supply, Burlington, NC) and maintained at 37°C in humidified 95% air and 5% CO₂ for 2 hours, and were studied no later than 8 hours after harvest.

L4&L5 DRGs from rats were rapidly harvested following isoflurane anesthesia and decapitation. Ganglia were placed in a 35 mm dish containing Ca²⁺/Mg²⁺-free, cold HBBS (Life Technologies) and cut into four to six pieces that were incubated in in 0.01% blendzyme 2 (Roche Diagnostics, Indianapolis, IN) for 26 min followed by incubation in 0.25% trypsin (Sigma Aldrich, St. Louis, MO) and 0.125% DNase (Sigma) for 30 min, both dissolved in Dulbecco’s modified Eagle’s medium (DMEM)/F12 with glutaMAX (Invitrogen, Carlsbad, CA). After exposure to 0.1% trypsin inhibitor and centrifugation, the pellet was gently triturated in culture medium containing Neural Basal Media A with B27 supplement (Invitrogen), 0.5mM glutamine, 10ng/ml nerve growth factor 7S (Alomone Labs, Jerusalem, Israel) and 0.02 mg/ml gentamicin (Invitrogen). Dissociated neurons were plated onto poly-L-lysine coated glass cover slips and maintained at 37°C in humidified 95% air and 5% CO₂ for 2 hours, and were studied no later than 6 hours after harvest.