1. Use day 9 chicken embryos.

2. Dissect sympathetic chains in Ham's F12 media.

3. Dissociate ganglia through incubation with 0.25% trypsin for 20 min at 37°C.

4. Wash tissue with 100% fetal calf serum for 5 minutes to end trypsinization

5. Wash chains 2x in Ham's F12 medium.

6. Dissociate tissue by gentle trituration with a flamed pipet

7. Plate onto pretreated (poly-dl-ornithine or poly-l-lysine) culture dishes containing 1.5 ml of either Neurobasal or serum-free F12+ medium

8. Add 10 ng/ml NGF

F12+ Medium contains these supplements:

- 100 µg/ml human transferrin

- 1 ml Pen/Strept/100ml

- 100 µM putrescine

- 20 nM progesterone

- 30 nM selenium

- 5 µg/ml bovine insulin