The Maintenance and Identification of Mouse Cerebellar Granule Cells in Monolayer Culture

Methods are described for maintaining postnatal mouse cerebellar cells in monolayer culture, and for identifying granule cells in such cultures. Cells from cerebella of 7-day-old mice are dissociated with trypsin and DNAse, then plated at 1-1.5 X 10(6) cells/35 mm dish in a high-potassium modification of Hams F12 medium plus 10% fetal calf serum. Under these conditions, cells grow either singly or in small clumps, and develop complex meshes of single fibers and fiber bundles over a period of several days. Granule cells are identified by a combination of several criteria including their size, shape and relative proportion of the total cell population as determined by phase contrast and scanning electron microscopy; nuclear morphology, demonstrated by transmission electron microscopy, and failure to take up [3H]gamma-aminobutyric acid (GABA) in the presence of several other cell types which do, shown by autoradiography.