Clonal analysis was conducted to assess whether aDRG NSCs could form neurospheres following dissociating the cells. We dissociated aDRG NSCs using 0.25% trypsin at 37°C for 30 min. Following three centrifuge–wash cycles, cells were plated in a 48-well culture dish at a final density of 10 cells/ml, in DMEM/F-12 medium plus 10 ng/ml EGF/bFGF, similar to methods previously reported for clonal neurosphere assays (36). EGF/bFGF supplementation was used based on optimal response as determined from results of trophic factor dependence. The number of cells and neurospheres in each well were counted at 7, 10, 14, and 21 days after plating. Neurospheres were defined as spherical-shaped aggregates containing five or more cells.
