Evaluating c-kit Expression in CSCs

The expression of c-kit in CSCs was evaluated by immunostaining as previously described [32 (link)]. In brief, twice-passaged CSCs (1×10⁴/well) were cultured in 8-well chamber culture slides (Lab-Tek, Thermo Scientific Nunc) coated with 15 μg/ml fibronectin for 3 days and then fixed with 4% PFA for 10 min. After blocking, the cells were incubated with PE-conjugated rat anti-mouse c-kit antibody (eBioscience). The nuclei were labeled with DAPI. The positively stained cells were counted under a fluorescent microscope with 200-fold magnification in twenty randomly selected fields, and the averages from 3 independent experiments were used for statistical analysis.