Protocol 52 Cell culture and RNA preparation

Cells were prepared and cultivated according to standard protocols from the literature. A detailed description is given in Gurok et al. (1). Briefly, the progenitor cells grew in suspension and formed spherical aggregates called neurospheres. Differentiation of neurosphere cells was induced by removing a mitogen epidermal growth factor (EGF), allowing the cells to attach to the dish which was coated with the adhesive substrate poly-L-lysine, and by addition of either brain-derived growth factor (BDNF) or neurotrophin 4 (NT4) to the differentiation medium. Both are neurotrophic factors and support neuronal differentiation. This treatment led to breaking up of the neurosphere aggregates. The cells migrated away from the sphere and changed their morphology (Figure 5.1). They also sythesized marker proteins for distinct neural lineages such as piii-Tubulin or GFAP (not shown).

We took samples of the cells before induction of differentiation and after 24, 48, and 96 h of differentiation. Total RNA was isolated with Trizol reagent (Invitrogen), precipitated with ethanol and resuspended in nuclease-free water. The concentration was determined spectrophotometrically and the quality was checked by agarose gel electrophoresis.

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