Info

Ingredient

Amount

Final concentration

DMEM 1 x (Gibco)

49 ml

49% (v/v)

F-12 nutrient mixture (HAM) 1 x (Gibco) 49 ml

49% (v/v)

Bovine serum albumin (BSA) (Calbiochem) 50 mg

0.5 mg/ml

200 mM L-glutamine (GibcoBRL)

1 ml

2 mM

N-2 supplement (Gibco)

1 ml

1 x or 1% (v/v)

Note: BSA is somewhat difficult to dissolve. Initially add the powder to 25 ml DMEM and then pipette up and down several times to dissolve. Combine with the other components and filter sterilize. Then add:

Nerve growth factor (Upstate) 100 ^l (from 100 ^g/ml stock) 100 ng/ml

Immediately divide into 10- or 30-ml aliquots in sterile polypropylene tubes and freeze at — 80 C for future use.

III. N2-Based Media For Long-Term Culture

Ingredient Amount Final concentration

Ingredient Amount Final concentration

N2 medium

99.2 ml

99.2% (v/v)

B-27 supplement (Gibco)

0.7 ml

1 x or 0.7% (v/v)

1-^-D-Arabinofuranosylcytosine

100 ^l (from 0.24 mg/ml stock)

0.24 ^g/ml

(Ara-C) (Calbiochem)

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