Cell culture
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DRGs from female Wistar rats (150 grams) were harvested in cold DMEM and any excess dorsal roots and spinal nerves were trimmed under a stereo microscope.
- DRGs were incubated with 0.125% Collagenase XI and 0.1 mg/ml DNase II in DMEM for 90 min at 37°C.
- After enzymatic digestion, DRGs were triturated with a cut 1 ml tip until a cell suspension was obtained.
- Cells were spun down, resuspended in pre-warmed DMEM and filtered through a 70 µm mesh .
- DRG neurons were recovered by using 10% BSA (PAA)/ DMEM cushions and plated on 13 mm glass coverslips coated with poly-L-lysine and laminin in complete media (DMEM, 10% fetal bovine serum (FBS), penicillin/streptomycin (1∶100), NGF (50 ng/ml) and aphidicolin (10 µM).
- 10,000 and 150,000 cells per coverslips were plated for immunofluorescence and biochemistry purposes, respectively.
- Cells were maintained in a 95% air/5% CO2 humidified incubator.
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NGF contents in the skin.
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NGF contents in the skin of the affected and contralateral hindlimbs of wild-type and Y1472F-KI mice on day 50 post-inoculation and in the back skin at 24 h after wounding were measured by ELISA.
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