Fig. 5. Pre-neoplastic cells can be isolated from the cerebellum of patched mutant mice. Cells were isolated from the cerebellum of wild-type and patched^+/– mice by enzymatic dissociation followed by Percoll gradient centrifugation, and viable cells were counted after Trypan Blue staining. (A) The average yield for 6-week-old wild-type mice was 0.53±0.25 million cells. For patched heterozygotes of the same age, the average yield was 4.3 million cells, with 84% of animals having more than 0.9 million cells (the maximum number seen in wild-type mice). (B) Among older patched mutants (10-25 weeks), 16% had tumors containing 50-600 million cells; the remainder had fewer than 2 million cells. (C) Non-granule cell precursors (GFP^- cells from neonatal Math1-GFP/patched^+/– mice), pre-neoplastic cells and tumor cells were stained with the fluorescent ß-galactosidase substrate FDG and analyzed by flow cytometry. Relative fluorescence of non-GCPs (blue), pre-neoplastic cells (pink) and tumor cells (purple) is shown. The horizontal black line indicates the range of fluorescence considered to be positive (i.e. above background); 89% of pre-neoplastic cells and 96% of tumor cells exhibited fluorescence within this range.