Tumorsphere formation correlates with presence of CD44+/CD24-/ESA+ cells. (a) Phase-contrast brightfield image of SUM159 tumorspheres at day 11 under a 10× objective. (b) Representative flow cytometry profiles of SUM159 cells grown in adherent or nonadherent cultures. Nonadherent tumorspheres were filtered through cell strainers to separate spheres smaller than 100 μm from spheres larger than 100 μm. Percentage indicated in epithelial-specific antigen (ESA) histograms represents the population of CD44+/CD24-/ESA+ cells. (c) For each cell line, 5,600 single cells/cm2 were plated on super-low adherence plates and spheres visible by eye were counted every 3 days. (d) The absolute number of tumorspheres on day 12 was divided by the corresponding crystal violet value (see Materials and methods) for each cell line to correct for the proliferation rate of the cell lines.