Neurobasal medium

Neuronal maintenance medium Neurobasal medium (GIBCO, Grand Island, NY), lx B27 (GIBCO, Grand Island, NY), 1 mM glutamine (GIBCO, Grand Island, NY). 

Medium 2 (from 4DIV, after transfection) 95 % Neurobasal Medium-A (Gibco ), 2 % B27 supplement 50x (Gibco ), 2 % 100 ruM L-glutamine, 1 % antibiotic/antimycotic solution. 

Maintenance medium Neurobasal medium supplemented with B27 (1X), L-glutamine (2 mM), andpenicillin/streptomycin antibiotics (all from Invitrogen ). 

Generally, a half bottle of the above medium is mixed with a half bottle of B27 supplement (50x, 10 ml, cat. 17504-044) and the Neurobasal culture medium is used within 2 weeks. The other half-bottle of B27 supplement will be kept frozen at —20° and thawed one more time for use in culture. If only a small quantity of culture medium is needed, aliquot the B27 supplement to 2 ml/tube, which is good for 100 ml of medium. 

After an hour of incubation, the medium is replaced with Neurobasal culture medium. Add 2, 4, and 10 ml of Neurobasal culture medium to the 35, 60, and 100-mm plates, respectively. After 5 to 7 days in culture, the medium must be half-changed, that is, about half of the old medium is replaced with the same volume of fresh Neurobasal culture medium. Halfchange the medium every 5 to 7 days until the neurons are ready to use. For neurons younger than DIV (days in vitro) 7, if necessary, the medium can be completely replaced without affecting cell viability. In contrast to Brewer s protocol (Brewer et ai, 1993), we do not include 25 /iM glutamate during the first four days of culture and find no adverse effect on neurons. 

The transfection mixture is then diluted into 1 ml of growth medium (Neurobasal media/B27) and mixed by pipetting up and down twice. 

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