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To obtain tumor spheres, cells were cultured in DMEM/F12 with 2% B-27 serum-free supplement (17504–044; Invitrogen), 20 ng/ml epidermal growth factor (EGF; PHG0311L; Invitrogen), and 20 ng/ml basic fibroblastic growth factor (FGF; PHG0266; Invitrogen) for 14 days to select for CSCs and early progenitor cells. Resulting tumor spheres were examined and counted under the microscope.
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