Correlation between KOr intensity and nestin expression levels or "stemness". (A) Dissociated neural cells were prepared from the cerebral cortices of E14.5 nestin/KOr embryos and immunocytochemically labeled for Nestin by using the same anti-Nestin primary Ab described in Figure 2 and an Alexa Fluor 488-conjugated goat anti-mouse secondary Ab (Jackson ImmunoResearch; 1:250). The processed cells were then analyzed for KOr and Alexa Fluor 488 fluorescence with a cell sorter (MoFlo; Beckman Coulter). (B) Dissociated neural cells were prepared as in (A) and sorted into four populations according to the intensity of the KOr fluorescence. About 500 cells of each population were plated per well at a density of 2.5 cell/μl in a 96-well plate for neurosphere formation assay. After 7 days of culture in medium containing EGF (BD Biosciences) and bFGF (Roche), the numbers of spheres in each well were determined.