Figure 2

4T1 ALDH ^br cells have high mammosphere-forming ability. (A) Representative flow cytometry plots demonstrating the sorting of 4T1 cells with high (ALDH^br, P2, 3~5% of P1 gate) and low ALDH activity (ALDH^lo, P3, 3~5% of P1 gate). The first gate (P1) chooses the cells with good status from the total cells and excludes cell debris according to their FSC & SSC values. The cells were incubated with an aldefluor substrate (BAAA), and the specific inhibitor of ALDH, DEAB, was first used to establish the baseline fluorescence level of ALDH activity (top). Staining of 4T1 cells with aldefluor substrate but without DEAB inhibitor produced a shift in BAAA fluorescence that defined the ALDH^br population (bottom). (B) ALDH^br (left) and ALDH^lo (right) cells were plated for mammosphere formation (5 × 10⁴ cells/well) for 8 days. Mammospheres were observed using an Olympus inverted phase contrast microscope (40 × objective magnifications). (C) The data shown are the average number of spheres counted in different sizes from a representative experiment performed in duplicate wells.