Flow cytometry analysis for cell cycle arrest

Cell cycle arrest was detected using flow cytometry⁶⁰. The cells were treated with the IC₅₀ concentrations of GT and FUM alone and in combination at 20% and 40% of the respective IC₅₀ concentrations, for 24 h, against A549 and L132 cells. The cells were harvested, washed with PBS, centrifuged at 1,000 rpm for 5 min, and fixed with 1 mL 70% ice cold ethanol overnight at 4 °C. Later, the ethanol was removed and 10 µL of RNase A (10 mg/mL) was added and incubated for 30 min. Again, cells were washed with PBS and re-suspended in 1 mL PBS with 50 µL Propidium Iodide (1 mg/mL) for 30 min in dark. The cells were analyzed using the BD FACSverse flow cytometer (Becton–Dickinson), and a peak fluorescence gate was used to discriminate the aggregates. A total of 20,000 events were acquired and three replicates were used per experimental condition. The percentage of cells in each phase was determined in reference to untreated cells.