Facsaria

The CNE-2s cells were placed in 6-well plates were starved for 6 h and treated with 1μM CQ and 10μM zinc for 24 h. The cells were harvested, washed twice with cold PBS, and stained with FITC-conjugated annexin V for 20 min and propidium iodide for 5 min (Sigma-Aldrich) in the dark. The stained cells were assessed by flow cytometry (FACSAria ^TM[Ⅲ], BD, Mountain View, USA)), and analyzed by FlowJo vX.0.7 software. The intracellular apoptosis rates were measured by flow cytometry as previously described 20 (link). Briefly, 510⁵ cells were seeded in 6-well plates and then treated as indicated. The cells were washed twice with cold PBS and collected for fluorescence analysis using a flow cytometer (FACSAria TM[Ⅲ], BD).

Mouse CD4⁺ T cells were sorted to ≥98% purity on a FACSAria (Becton Dickinson, Oxford, UK). Human CD4⁺ T cells were negatively selected to >90% purity from peripheral blood mononuclear cells (PBMCs) by removing CD8⁺, CD25⁺, CD56⁺, CD14⁺, and CD19⁺ populations (FACSAria; Becton Dickinson).

For single-cell cloning, clonally barcoded MDA-MB-157 cells were trypsinized, washed with PBS supplemented with 3% FBS, sent through a 40 µm filter, and resuspended to 1 × 10⁶ cells ml⁻¹. A FACSaria (Becton Dickinson) was used to sort single cells into wells of 96-well plates, each well containing 100 µl of DMEM with 10% FBS.
After expansion of the barcoded population of cells, a portion of the cells were stained for keratin 8/18 expression and sorted via FACS. Portions were separated out of the population and sorted at three time points each separated by a week (day 0, day 7, day 14).
For these sorts based on keratin 8/18 expression, cells were stained as in intracellular flow cytometry, but stained at 1 × 10⁷ cells ml⁻¹ in FB and with a 1 : 60 dilution of mouse anti K8/18, clone C51 (Cell Signaling), for 1–2 h on ice. After secondary staining and washes, cells were resuspended at 1 × 10⁷ cells ml⁻¹ in FB and sorted on a FACSaria (Becton Dickinson) set to maximize yield. Sorted samples were analysed on the FACSaria to measure the proportion of cells mis-segregated, counted on a haemocytometer, and split in half. Barcodes were extracted from these cells as described below.

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.