Preparation of primary rat DRG cells
DRG cells were isolated according to the methods described byBurkey et al. (Burkey & Hingtgen et al., 2004), with some modification. Healthy Sprague-Dawley rats (male, ~250 g) were placed on the operating table after ether anaesthesia and disinfected with 75% ethanol. The spinal canals were separated, and the DRGs were collected in cold Hank’s balanced salt solution (HBSS). All operations were performed quickly on ice. After digestion with 1.25 mg/mL collagenase IV (90 min, 37 °C) and 0.025% trypsin (90 min, 37 °C), the DRG cells were dispersed in a medium containing 50 μM 5-fluro-2′-deoxyuridine. Sensory neurons were plated at 28,000–30,000 cells per well in 6-well tissue-culture plates or 7500-8000 cells per well in 96-well tissue-culture plates. The cells were maintained at 37 °C under a 5% CO2 atmosphere.