Slowly dividing CD44+/Flk-1+ cells show characteristics of stem-like cancer cells. (a) Hoechst exclusion assay. Vybrant CM DiI-labeled cells were seeded at clonal density and allowed to propagate until reaching 70% confluence. Hoechst exclusion (arrows) was only found in slowly dividing cells that retained Vybrant CM-DiI staining. Scale bar = 5 μm. (b) Cells were cultivated as described for panel a and then FACS sorted for high Vybrant CM-DiI staining. After re-cultivation of this cell fraction, Hoechst exclusion (arrows) was again found only in cells that retained Vybrant CM-DiI staining. Scale bar = 5 μm. C. Immunocytochemistry for CD44 and CD24. Note that CD24 shows background expression and that CD44 and Vrybrant CM-DiI are co-distributed (purple in overlay). Scale bar = 20 μm. FACS, fluorescence-activated cell sorting.