Isolation and Culture of Mouse Cortical Astrocytes

The overall aim of this procedure is to obtain pure astrocyte cultures by isolating and culturing mixed cortical cells from P one to P four mouse pups. This is accomplished by first harvesting brain cortices of mouse pups and removing the meninges. The second step is to cut the cortex into small pieces, dissociate the cortex pieces by trypsin and tation to obtain single cells and to plate the single cells on poly de lycine coated tissue culture flasks.

Next, after seven to eight days, astrocytes, microglia, and oligodendrocytes will have formed different layers and the separation of astrocytes from microglia and oligodendrocytes is achieved by shaking the tissue culture flask on an orbital shaker. The final step is the ionization and harvest of the remaining astrocytes, which are then replated in a tissue culture flask. Ultimately 12 to 14 days after the first cell split, astrocytes are plated at the appropriate concentration for experiments and cell purity can be examined by immuno cyto chemistry using astrocyte specific markers.