ALDEFLUOR assay and separation of the ALDH positive population

ALDH activity was assessed by FACS analysis in MCF7 cells cultured for 72 h in Normoxia (21% O2) or Hypoxia (1% O2), as well as in MCF7 cells treated with Vehicle or Valproic Acid for 72 h. The ALDEFLUOR kit (StemCell Technologies) was used to isolate the population with high ALDH enzymatic activity by FACS (Fortessa, BD Bioscence). Briefly, 1 × 105 MCF7 cells were incubated in 1ml ALDEFLUOR assay buffer containing ALDH substrate (5 μl/ml) for 40 minutes at 37°C. In each experiment, a sample of cells was stained under identical conditions with 30 μM of diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, as negative control. The ALDEFLUOR-positive population was established in according to the manufacturer’s instructions and was evaluated in 3 × 104 cells. Data analysis was performed using FlowJo software (Tree star Inc.).