Facs calibre

Cell proliferation was performed using CellTiter 96 Aqueous One Solution Cell Proliferation Assay according to the manufacturer's instruction (MTS Assay, Promega, WI, USA). For this assay, 5,000 cells were cultured per well in a 96-well plate. Transfection of microRNA mimics were performed at the same time (measured as day 0) and proliferation data, using absorbance was measured on day 1 to day 5 following transfection. Absorbance at 490 nm was measured using a 96-well plate reader (Sunrise microplate reader, Tecan, Switzerland). For cell cycle analysis, 2.5 × 10⁵ H295R or SW-13 cells were cultured in a 6-well plate. Cells were collected, washed with Phosphate Buffered Saline (PBS) and stained with Propidium Iodide (PI, Sigma Aldrich) at a final concentration of 17.4 μg/ml. Cells were analysed using fluorescence-activated cell sorting (FACS) analysis (FACS Calibre, BD Biosciences) and flow cytometry histograms were modelled using Modfit LT software (Verity Software House, ME, USA).

MCF-7 cells were incubated at a density of 4 × 10⁶/well with synthesised compounds 6a and 6i at their IC₅₀ for 24 h. Cells underwent three washing cycles using ice cold PBS, then suspended in PBS. Cell apoptosis was detected by Annexin V-FITC Apoptosis Detection Kit (BioVisionResearch Products, USA). The cells were stained using PI staining solution, Annexin V-FITC and incubated for 15 min at room temperature in a dark place. Cells were investigated by flow cytometry (Ex = 488 nm; Em = 530 nm) using FACS calibre (Becton Dickinson, Heidelberg, Germany)⁴⁸ .

For direct detection of cell surface proteins, cells were incubated with fluorescently labelled antibodies for 30 mins at 4°C or 15 mins at room temperature. Labelled cells were washed twice with FACS wash (PBS, 1% w/v BSA (Europa Bioproducts), 0.01% v/v sodium azide (Sigma-Aldrich) and centrifuged at 300 × g for 5 mins. For indirect detection of hIgG bound to the cell surface, target cells were opsonised with mAb for 30 mins at 4°C or 15 mins at room temperature, washed twice with FACS wash, 0.01% sodium azide (Sigma-Aldrich) at 300 × g for 5 mins, and labelled with mouse anti-human IgG-APC (clone M1310G05) for 30 mins at 4°C or 15 mins at room temperature. Labelled cells were washed twice with FACS wash at 300 × g for 5 min. Samples were analysed using a FACS Calibre or Canto (Becton Dickinson) and data analysis was performed using FlowJo (Becton Dickson).

Cell apoptosis assays were performed using a BD FACS Calibre flow cytometer (BD, New York, NY, USA). Briefly, 1 × 10⁵ cells were seeded in a 10‐cm dish containing a regular complete medium. After 6 h, cells were treated with either NSC‐CM or control NSC culture medium for various durations. The cells were then harvested, washed in PBS and stained using an APC Annexin V Apoptosis Detection Kit with PI (BioLegend, San Diego, CA, USA) for 10 min. Early and late apoptotic cells were quantified using a flow cytometer.

NHS was prepared from the blood of healthy volunteers with appropriate consent. Venous blood was taken into glass vials to clot. Clotted blood was centrifuged at 900 × g for 20 mins and collected serum stored in glass vials at −80 °C. For the CDC assay, CD20+ Ramos or Raji cells were opsonised with mAb at the desired concentrations for 30 mins at 4°C. NHS was then added at 20% V/V and incubated for 30 mins at 37°C. Cell death was measured as propidium iodide (PI) positive cells (%) by flow cytometry (BD FACS Calibre).