2.1.1 Cell culture:
60 male Wistar rats (200–300g) were killed by stunning and cervical
dislocation under Schedule 1 according to the United Kingdom Animals
(Scientific Procedures) Act 1986. DRGs were removed from male Wistar
rats and collected in cold (4°C) (HBSS; Gibco BRL). Using sterile
scissors, the DRG were chopped into small fragments to increase surface
area for enzyme digestion. Ganglia were incubated in (papain (1mg/ml)
and L-cysteine (2mg/ml) in HBSS (Gibco BRL) for 15 min at 37°C. Ganglia
were then replaced in (dispase (0.8%) and collagenase type F (1mg/ml,))
in HBSS for 22-30 min at 37°C, during the incubation a mechanical
trituration was performed by using a sterile, fire-polished glass
Pasteur pipette to enhance the disaggregation of neurons in the clamp.
the cell suspension was centrifuged for 5 min at 1000g at 4°C this was
then followed by the resuspension of the cell pellets with sterile HBSS
to remove the enzyme. Cell pellets were resuspended in 1ml of feeding
media, and the DRG neurons were plated onto the 16mm coverslips
(Scientific Laboratory Supplies) coated with poly-D-lysine (0.1mg/ml,
Sigma Aldrich) and laminin (20µg/ml, Sigma Aldrich). Cultures were
maintained for 2d in feeding medium containing Ham’s F-12 supplemented
with 10% horse serum (Sigma Aldrich), 2mM glutamine (Sigma Aldrich),
10ng/ml nerve growth factor (NGF) (Sigma Aldrich),and 1%
penicillin/streptomycin (enzyme activity = 5,000units/ml penicillin
and5,000µg/ml streptomycin, Gibco at 37°C in humidified air with 5%
CO2.