Facs calibur 4

To validate our AO/PI data, 3,4-diarylpyrazoles (50 μM) treated MDA-MB-231 cells were stained with Annexin V/PI and FACS analysis were carried out. In detail, after treatment, the MDA-MB-231 cells were incubated with 2μl of annexin V-FITC and 5μl of PI (100 μg/ml) in 200μl of binding buffer at room temperature for 15 min in the dark. After washing in 1X PBS, stained cells were examined by flow cytometry using BectoDickinson fluorescence-activated cell sorter (FACS) IV Calibur (Becton Dickinson, San Jose, CA, USA). The flow cytometry data were analyzed and plotted for annexin V-FITC using a Cell Quest software (Becton Dickinson).

Flow cytometric analyses were performed using a standard protocol. Induced GSC-like cells, dissociated in trypsin-EDTA, and aliquots of 10⁶ cells were resuspended in 0.1 ml of PBS containing 2% fetal bovine serum (PBS/FBS) and subsequently incubated with primary antibodies. To quantify GSC-like cells, the cells were incubated with 10 mg/ml of GFR-α1 antibody (Chemicon). The primary antibodies were detected using 5 mg/ml of a FITC-conjugated secondary antibody. The control cells were not treated with primary antibodies. The cells were maintained in the dark on ice until analysis on a Becton Dickinson FACS IV Calibur (Becton Dickinson, San Jose, CA). At least 5000 to 10000 events were acquired for each sample.
To analyze the DNA content after germ cell differentiation, the encapsulated cells were dissociated in trypsin-EDTA and resuspended in DPBS to release single cells. After filtering through 80-µm nylon mesh, the cells were fixed in cold 70% ethanol and maintained at 4°C until further analysis. For the DNA content assay, 1×10⁶ cells were washed twice with DPBS and incubated in 500 µL of 0.2% pepsin for 10 min at 37°C. After centrifugation, the cells were stained in a solution containing 25 µg/ml propidium iodide (PI) (Sigma), 40 µg/ml RNase (Gibco/Invitrogen), and 0.3% Tween-20 in PBS at room temperature for 20 min. The stained cells were analyzed using a FACS IV system.