Cell surface expression of ESA, CD44, and CD24. Three-color flow cytometry analysis was performed to detect the CD44+/CD24-/ESA+ cell population. (a) Top panel: Unstained, isotype control, and ESA-stained cells are shown in the histogram. ESA staining of MDA-MB 231 (total cell population) and MDA-MB 231 F3 (cycling hypoxia-selected subpopulation) cells. M1 marker gates ESA+ cells, and M2 marker gates the ESA- cells. Bottom panel: CD44 and CD24 expression of ESA+ cells in each cell line. The percentages of the CD44+/CD24- and CD44+/CD24+ cells within the ESA+ cell population are indicated in the Quad plot. (b) The same flow cytometry analysis for BCM2 (total cell population) and BCM2 F3 (cycling hypoxia-selected subpopulation) cells. (c) Quantitative comparison of CD44+/CD24-/ESA+ cell population and CD44+/CD24+/ESA+ cell population in cycling hypoxia-selected subpopulations (MDA-MB 231 F3 and BCM2 F3) and their parental breast cancer cell lines. At least three replications were performed to derive the average percentage of each cell population in each cell line and standard deviations. Asterisks indicate statistical significance by two-tail t test (n = 3, P < 0.05). ESA, epithelial-specific antigen.