2.11 Preparation of DRG neuronal cultures
Primary cultures of DRG sensory neurons were obtained from naïve,
3–4-week-old, male C57Bl/6 and Oprm1 cKO mice weighing 12–15 g.
Once the mice were anesthetized, the DRGs were carefully removed and
immediately placed into fresh, ice-cold DH10 media consisting of
DMEM/F-12 supplemented with 10% fetal bovine serum (no DMSO) and 1%
penicillin/streptomycin (~1.5 ml per animal), washed
with Hanks’ balanced salt solution (HBSS) 1–2×, and then treated with
an enzyme solution containing 5 mg∙ml-1 dispase and 1
mg∙ml-1 collagenase type I in HBSS without
Ca2+ and Mg2+ (Gibco). Cells were
shaken slowly for >45 min at 37°C. After trituration
(10–15×) and centrifugation, the cells were resuspended in DH10 media
containing nerve growth factor (50 U∙ml-1; Upstate
Biotechnology) and plated on freshly made poly-D-lysine- (100
µg∙ml-1; Biomedical Technologies) and laminin- (100
µg∙ml-1) coated coverslips. Cultures were then
incubated at 37°C in 5% CO2 until being used in
subsequent electrophysiological studies.