Flow cytometry and ALDEFLUOR assay

1 × 10⁶ cells were harvested and washed in PBS three times. Then, the cells were resuspended in 100 μl Flow Cytometry Staining Buffer (eBioscience). Two microliter of CD133 (STEM CELL Technologies Inc, Canada) were added and incubated for 20 min in dark. Next, the cells were washed by Flow Cytometry Staining Buffer and prepare to be detected. The CD133-positive subpopulation was detected by FACS (BD Accuri C5, USA).

For the ALDH assay, cells were identified using the ALDEFLUOR reagent kit (STEMCELL). Cells were suspended in ALDEFLUOR assay buffer at a concentration of 1 × 10⁵ cells/ml and divided into two tubes labeled “control” and “test”. Diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, was added to the control tube to control for background fluorescence. Activated ALDEFLUOR reagent was added to tubes. The tubes were incubated for 30 min at 37 °C, and the tubes were then centrifuged for 5 min at 250 × g. Cell pellets were resuspended in ALDEFLUOR assay buffer and stored on ice. The ALDH1-positive subpopulation was detected by FACS (BD Accuri C5, USA).