Aldefluor assay

The Aldefluor assay (Stem Cell Technologies) was used to identify and isolate cell populations with ALDH1 enzymatic activity. Previous studies in our laboratory have characterised ALDH1+ve cells as CSCs, thus providing the foundation on which this CSC study has been carried out [27]. The assay was performed according to manufacturer's instructions. Briefly, cells (5 × 10⁵) were suspended in Aldefluor assay buffer containing activated Aldefluor reagent, BODIPY-aminoacetaldehyde (BAAA) for 45 min. This is a fluorescent non-toxic ALDH1 substrate that freely diffuses into intact viable cells. In the presence of ALDH1, BAAA is converted to BOPIDY-aminoacetate (BAA) which is retained within the cells expressing ALDH1. A specific ALDH1 inhibitor, DEAB, was used to inhibit the BAAA-BAA conversion and acts as an internal negative control for background fluorescence. The brightly fluorescent ALDH1+ve cells were detected using the green fluorescence channel (520–540 nm). ALDH1 activity was measured using a CyAn™ ADP flow cytometer (Dako, USA), while ALDH1+ve and ALDH1-ve fractions were sorted using a MoFlo™ XDP high speed cell sorter (Beckman Coulter, USA). For FACS analysis of ALDH1, gates were set for each sample relative to their ALDH1 inhibitor (DEAB) controls. The DEAB control is required for accurate compensation of fluorescent signals and for setting appropriate gates to discriminate between ALDH1+ve and ALDH1-ve cells in addition to background autofluorescence which varied across all parental and corresponding cisplatin resistant cell lines.