CD44+/CD24-/ESA+ cells retain BrdU label. (a) SUM159 were continuously treated with 5-bromo-2-deoxyuridine (BrdU) for 10 days. Cells that stain for BrdU label 6 days after chase exhibit either a solid or speckled pattern of label retention: BrdU (green) or DAPI (4',6 diamidino-2-phenylindole; blue). (b) SUM159 and MDA.MB.231 cells were pulse-chased with BrdU. The percentage of cells with either solid or speckled BrdU staining were counted at the indicated time points. Data are presented as the mean ± standard error of the mean for at least three separate 10× fields of two separate experiments. (c) Proliferating cultures of SUM159 cells were pulse-chased and analyzed by flow cytometry for CD44+/CD24-/ESA+ and the presence of BrdU. Note that about 90% of CD44+/CD24-/ESA+ SUM159 cells retain BrdU label at 6 days after chase. (d) Immunoflourescence of BrdU and Ki67 in cells that were BrdU pulse-chased. (i) Co-localization of BrdU (green) and Ki67 (red); note that of the adjacent nuclei that are both Ki67 positive, only one contains BrdU. ESA, epithelial-specific antigen.