ALDEFLUOR assay

An ALDEFLUOR kit (StemCell Technologies) was used to isolate ALDEFLUOR⁺/ALDH1^high CSCs and ALDEFLUOR^-/ALDH1^low non-CSCs from patient-derived tumor cells or SW480 cells. A fluorescence substrate (BAAA), which can freely diffuse across cell membranes and be oxidized by ALDH1, was used in the ALDEFLUOR experiment. The oxidized BAAA provided a bright fluorescence signal that could be recognized by the flow cytometer. For each independent assay, diethylaminobenzaldehyde (DEAB) was used as an ALDH1 inhibitor to stain the cells as a negative control. Flow cytometry sorting gates were established by excluding propidium iodide-positive cells and ALDEFLUOR-positive cells after DEAB treatment.