(A) ALDEFLUOR FACS analysis of the breast cancer cell line MDA-MB-468. Cells incubated with ALDEFLUOR substrate and the ALDH-specific inhibitor DEAB were used to establish baseline fluorescence (ALDH1^–) and to define the ALDH1⁺ region (ALDH1⁺) (left panel). Incubation of MDA-MB-468 cells with ALDEFLUOR substrate in the absence of DEAB induced a fluorescence shift defining the ALDH1⁺ cell population (right panel). (B and C) ALDH1⁺ cell populations (top 10% of cells with high fluorescence) and ALDH1^– cell populations (bottom 10% of cells with low fluorescence) were sorted from MDA-MB-468 cells. Sorted ALDH1⁺ and ALDH1^– cells were measured for mammosphere formation (B) and K353 acetylation of ALDH1A1 (K353Ac) (C). Relative K353 acetylation indicates the intensity ratio of K353Ac/ALDH1A1 protein levels. (D) Representative FACS analysis of primary breast cancer cells by ALDEFLUOR assay. (E and F) ALDH1⁺ cell populations (top 3% of cells with high fluorescence) and ALDH1^– cell populations (bottom 10% of cells with low fluorescence) were sorted from primary breast cancer cells, followed by a mammosphere-forming assay (E) and Western blotting with the indicated antibodies (F). (B and E) Data represent the mean ± SD of triplicate experiments for the number of mammospheres per 10,000 transplanted cells.