Isolating ALDH-high Cancer Cells

The ALDEFLUOR kit (STEMCELL Technologies, Canada) was used to isolate the population with a high ALDH enzymatic activity [8 (link)]. HCC1937/p53 cells were suspended in the ALDEFLUOR assay buffer containing ALDH substrate (BAAA, 1 μmol/L per 1 × 10⁶ cells) and incubated at 37°C for 30 min. In each experiment, a sample of cells was incubated with 50 mmol/L of the specific ALDH inhibitor diethylaminobenzaldehyde (DEAB) as a negative control. Flow cytometry sorting was conducted using a Cell Sorter SH800 (SONY, Tokyo, Japan) and SH800 Software (Ver. 2.1.2) (SONY, Tokyo, Japan). ALDEFLUOR fluorescence was excited at 488 nm wavelength and fluorescence emission was detected using a standard FITC 525/50 band pass filter (FL2). The sorting gates were established by distinguishing viable from nonviable cells using 7-aminoactinomycin D (7-AAD) (BD Biosciences, USA). The data were analyzed using SH800 Software (Ver. 2.1.2) (SONY, Tokyo, Japan).