Poly d lysine

Cells were plated in poly D-Lysine coated 96-well plates on day 0 (Becton Dickinson Labware). On day 1, the indicated medium was added, and measurements were performed on day 2. For Kir4.1 experiments, stably transfected cell lines were made by subcloning wild-type or mutant Kir4.1 into pIRES-puro3 (Clontech) and selecting cells with puromycin (1 ug/ml). Stable cells were plated in normal K⁺ Dulbecco’s modified Eagle’s medium (DMEM), and Cl⁻ was measured. Additional details are provided in the Supplemental Information.

Mice were anesthetized with isoflurane and sacrificed by decapitation and the TG or dura mater were removed and prepared for culture as previously described (24 (link)). For cultures with back-labelled TG neurons, TGs were taken 7 days following supra-dural application of the retrograde tracer, wheat germ agglutinin conjugated Alexa-Fluor 555 nm. After removal, tissue was placed in ice-cold Hanks balanced-salt solution (divalent free). Trigeminal tissue was dissociated enzymatically with collagenase A (1 mg/ml, 25 min, Roche, Indianapolis, IN) and collagenase D (1 mg/ml, Roche, Indianapolis, IN) with papain (30 units/ml) for 20 min at 37°C. Dural tissue was dissociated enzymatically with collagenase A (1 mg/ml, 30 min, Roche, Indianapolis, IN) and collagenase D (1 mg/ml, Roche, Indianapolis, IN) with papain (30 units/ml) for 25 min at 37°C. The tissues were then triturated through fire-polished Pasteur pipettes, and trigeminal cells were plated on poly-D-lysine (Becton Dickinson) and dural cells plated on untreated plates. After several hours at room temperature to allow adhesion, cells were placed in a room-temperature, humidified chamber in Liebovitz L-15 medium supplemented with 10% FBS, 10 mM glucose, 10 mM HEPES and 50 U/ml penicillin/streptomycin. Trigeminal cells were tested within 24 hours post plating and dural cell cultures were tested 2–4 days post plating.

Seven days following fluorogold application, trigeminal ganglia (TG) were removed, enzymatically treated, and mechanically dissociated as previously described [36 (link); 39 (link)]. Rats were anesthetized with isoflurane (Phoenix Pharmaceuticals) and sacrificed by decapitation. The TG were removed and placed in ice-cold Hanks balanced-salt solution (HBSS, divalent free). Ganglia were cut into small pieces and incubated for 25 min in 20 U/ml Papain (Worthington) followed by 25 min in 3 mg/ml Collagenase Type II (Worthington). Ganglia were then triturated through fire-polished Pasteur pipettes and plated on poly-D-lysine (Becton Dickinson) and laminin (Sigma)-coated plates. After several hours at room temperature to allow adhesion, cells were cultured in a room-temperature, humidified chamber in Liebovitz L-15 medium supplemented with 10% FBS, 10 mM glucose, 10 mM HEPES and 50 U/ml penicillin/streptomycin. Cells were used within 24 h post plating.