Isolation and Characterization of Ovarian Cancer Stem Cell Subpopulations

FACS was performed according to the method described previously [10 (link)] with suitable modifications. Cell line single-cell suspensions were counted and incubated with CD133 primary antibodies, and then ALDH⁺ enzymatic activity was defined using the ALDEFLUOR kit per the protocol (Stem Cell Technologies, Vancouver, BC, Canada). For each sample, half of the cell/substrate mixture was treated with 50 mmol/L diethylaminobenzaldehyde (DEAB). Cells were incubated for 45 min. Gating for viability was established using propidium iodide (PI) exclusion and ALDEFLUOR/DEAB-treated cells were used to define negative gates. FACS was performed with ≥1 × 10⁵ cells using the BD FACSCanto II (Becton Dickinson, Franklin Lakes, NJ, USA) or FACSAria (Becton Dickinson) under low pressure in the absence of UV light. In all experiments, the ALDEFLUOR-stained cells treated with DEAB served as ALDH-negative controls. The ALDH⁺CD133⁺, ALDH⁻CD133⁺, ALDH⁺CD133⁻, and ALDH⁻CD133⁻ subpopulations were separated from the A2780 ovarian cancer cells by a FACSAria (Becton Dickinson). After sorting, all the cell subpopulations were cultured in a RPMI-1640 basic culture medium for 2 h; then, the cells were treated with different nanomaterials such as GO (50 μg/mL), rGO (20 μg/mL), rGO-Ag nanocomposite (10 μg/mL), and AgNPs (15 μg/mL) for 24 h.