Differentiate ES cells into glial cells and neurons

Day -1: Pass ES cells at normal density on gelatinized plate to free the culture of contamination fibroblast cells.

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Day 1 : Trypsinized the cells as for normal passaging until the colonies lift off. Try to keep the loosely connected clumps of cells together by gentle handling. Then directly plate the cells 1:3 into bacterial grade Petri dishes in LIF free medium containing 1 microM all-trans retinoic acid (RA).

Day 3 : Collect cell aggregates and replate in tissue culture dishes (appr. 25 cell aggregates per 6 cm tissue culture dish) in ES cell culture medium without LIF and RA. Aspirate the medium carefully.

Day 8 : Change half of the medium. From this day on at least 10% of the cells exhibits neuronal phenotype. They are specifically stained with Cresyl Violet and stongly positive for the N-CAM antigen.