Primary Culture of Adult Neural Progenitors - Drugs of Abuse: Neurological Reviews and Protocols - page 388

3. Dissecting microscope (Leica).

4. Artifi cial cerebrospinal fl uid (aCSF): 26 m M NaHCO 3 , 124 m M NaCl, 5 m M
KCl, 2 m M CaCl 2 , 1.3 m M MgCl 2 , 10 m M D -glucose, 100 U/mL of penicillin,
100 µ g/mL of streptomycin, pH 7.3.

5. Surgical instruments for microdissection (scalpel, dissecting scissors, and needle
probes).

6. Dissociation media: aCSF containing high Mg 2+ and low Ca 2+ (26 m M NaHCO 3 ,
124 m M NaCl, 5 m M KCl, 0.1 m M CaCl 2 , 3.2 m M MgCl 2 , 10 m M D -glucose,
100 U/mL of penicillin, 100

g/mL of streptomycin, pH 7.3). This solution can
be aliquoted and frozen at -20°C. Add 0.2 mg/mL of kynurenic acid (Sigma) before
use. Add 1.33 mg/mL of trypsin (Sigma) and 0.67 mg/mL of hyaluronidase
(Sigma) before use.
7. Hybridization oven.
8. Polypropylene 15-mL and 50-mL capped tubes (VWR, Mississauga, ON).
9. 35-mm, 100-mm diameter, and 12-well tissue culture dishes with transwel
inserts (VWR).
10. Enzyme inactivation media: Neurobasal media, 2 m M glutamine, 10% fetal calf
serum, 10% horse serum, 100 U/mL of penicillin, 100

µ

µ g/mL of streptomycin

(Invitrogen, Burlington, ON).
11. 30-mL syringe and 18-gauge needles.
12. Maintenance media: Neurobasal media, 2 m M glutamine, 25 mg/mL of insulin
(Sigma), 100 mg/mL of transferrin (Sigma), 20 n M progesterone (Sigma),
60 M putrescine (Sigma), 30 m M sodium selenite (Sigma), 20 ng/mL of

epidermal growth factor (Invitrogen), 10 ng/mL of basic fi broblast growth factor
(Invitrogen).
13. Trypan blue stain (Invitrogen).
14. Orbital rotator/shaker.
15. Vortexer/mixer.
16. LabTek II Chamber Slide System (VWR).
17. Phosphate-buffered saline (PBS): 10 m M sodium phosphate, 154 m M NaCl.
18. Poly- D -lysine (1 mg/mL, Sigma) in double-distilled H 2 O (ddH 2 O). Dispense into
1-mL aliquots. Store at -20°C.
19. Poly- D -lysine/laminin coating solution (100

µ

g/mL of poly- D -lysine, 0.02 mg/mL
of laminin). Add 1 mL poly- D -lysine stock (1 mg/mL) and 400

µ

L of 0.5 mg/mL
of laminin (Invitrogen) to 8.6 mL of sterile ddH 2 O. Use immediately.
20. Differentiation media with serum: Neurobasal media, 2 m M glutamine, 5% fetal
calf serum, 5% horse serum.
21. Differentiation media without serum: Neurobasal media, 2 m M glutamine,
5 mg/mL of insulin (Sigma), 100 mg/mL of transferrin (Sigma), 20 n M proges-
terone (Sigma), 60

µ

M putrescine (Sigma), 30 m M sodium selenite (Sigma),
1X B27 Supplement (Invitrogen).
22. Brain-derived neurotrophic factor (Invitrogen).
23. Matrigel (Becton Diskinson, Bedford, MA).

µ