Info

an = Number of dishes in which cell counts were taken in 1-mm circles etched shortly after plating [see Heidemann et al. (2001) for more on the method of quantitation of development]. All media contained 10% fetal bovine serum and the N9 supplement.

an = Number of dishes in which cell counts were taken in 1-mm circles etched shortly after plating [see Heidemann et al. (2001) for more on the method of quantitation of development]. All media contained 10% fetal bovine serum and the N9 supplement.

development than Medium 199 (Table I) and cultures stay healthy only for 5-7 days. However, this medium is clearly superior to the use of HEPES buffering in any of the aforementioned CO2-dependent media or CO2-independent medium (Life Technologies Inc.). L15 would appear to be advantageous for undergraduate laboratories in that it requires only a space heated to 37° C, not the CO2 tank, regulator, and incubator typically used. Also useful for the contraints of undergraduate laboratories, respectable axonal development (15% in stage 3 at day 3) can be achieved with only L15+ or Medium 199 and serum without the additional supplements outlined later.

Our current optimal conditions for growth and development include a large number of supplements, but which we have combined so that culture preparation is not onerous. We prepare both media from dry powder to which we add 100 U/ml of penicillin and 100 yg/ml streptomycin. We also add 0.6% glucose and 2 mM glutamine to L15 (Bray, 1991), which we call L15+. Just prior to use, mix either L15+ or Medium 199 with three additional liquid components: (a) 10% fetal bovine serum (horse serum does not support robust axonal development), (b) 7S nerve growth factor (100 ng/ml from a 1-mg/ml stock, Harlan Bioscience Inc.,

Table II

Preparation of N9 Neuronal Growth Supplement

A. Individual component stock solutions

Selenium dioxide0 37.5 ^g/ml

Putrescine dihydrochloride 16.1 mg/ml in phosphate-buffered saline (PBS) in PBS

A. Individual component stock solutions

Selenium dioxide0 37.5 ^g/ml

Putrescine dihydrochloride 16.1 mg/ml

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