Primary neuronal cultures
Cortices and hippocampi were isolated on embryonic day 16 (E16.5) rinsed
and dissected in ice-cold PBS and incubated with 0.25 % trypsin for 25
min at 37 °C. The digestion was terminated by the addition of DMEM
solution supplemented with 10 % FBS followed by trituration tissue
dissociation. The resulting cells were centrifuged for 5 min at 800 rpm
and neurons dissolved in Neurobasal medium supplemented with 2 % B-27,
0.5 mM L-glutamine and 1 % penicillin/streptomycin. The cells were
plated at a density of 2x105 cells/well in 6-well
poly-L-lysine-coated tissue culture dishes or on coverslips where
necessary. Cells were cultured for 10 days (DIV10) for neuron maturation
under 5 % CO2 at 37°C. Neuronal purity was
>90 % as determined by immunofluorescence using the
neuronal marker βIII-tubulin.