Aldefluor

ALDH enzymatic activity was assessed as previously described [34 (link)–36 ], using ALDEFLUOR (Stem Cell Technologies, Seattle, WA) according to the manufacturer’s instructions. Following ALDH staining, cells were incubated with CD133-APC antibody (BD Biosciences, Ashland, OR) at 1:20 dilution in ALDEFLUOR buffer for 25 min on ice, protected from light. Cells were washed in PBS and resuspended in 400 µl PBS for analysis on a BD FACSVerse cell analyzer (BD Biosciences, Franklin Lakes, NJ). Cell death was assessed by Annexin V (640,905, Biolegend, San Diego, CA) and propidium iodide (PI) (R37169, Thermo Scientific, Waltham, MA) staining on cells treated as indicated, as previously described [35 , 36 ].

Cells were pretreated and dissociated. For evaluation of the tumor-initiating feature, they were stained with ALDEFLUOR (STEMCELL Technologies, Vancouver, Canada) in presence or absence of the inhibitor diethylaminobenzaldehyde (DEAB; STEMCELL Technologies, Vancouver, Canada) to detect the specific marker ALDH [65 (link)]. In brief, cells were mixed in buffer-diluted ALDEFLUOR, and for control, was immediately transferred to DEAB. After incubation in 37 °C for 30 min, the cells were washed and resolved in the buffer. For ROS, they were labeled with dihydroethidium (Cayman, Ann Arbor, MI, USA). After incubation in 37 °C for 30 min, they were washed and resolved in PBS. The staining results of these cells were then studied and quantified for up to 10,000 cells using fluorescence-activated cell sorting (FACS) with a FACSCalibur system (BD Biosciences, Franklin Lakes, NJ, USA). The data were then analyzed via CellQuest (BD Biosciences, Franklin Lakes, NJ, USA).
The sorting process was through FACSAria™ III (BD Biosciences, Franklin Lakes, NJ, USA) for concomitant isolation of the ALDH-positive and the -negative cells, according to the ALDEFLUOR staining result. The cells were collected in the serum-free, growth factor-supplemented DMEM/F12 medium. At least 20,000 cells were used in each group for immediate RNA preparation, followed by the qPCR as described previously.