Annexin 5 and dead cell reagent

The GBM8401 and A172 cells were plated in six-well plates, with each well containing 500,000 cells. The cells were cultivated for 72 h to reach logarithmic phase growth. Following this, the cells were treated with varying concentrations of Arylquin 1 (0, 1, 2.5, and 5 µM) in complete culture medium for another 72 h. Subsequently, 1 million cells from each treatment group were isolated and resuspended in 100 µL of Annexin V and dead cell reagent (Merck Millipore, Warsaw, Poland) for 20 min at ambient temperature, using the medium as a control group. The apoptosis levels were quantified using a Muse^® Cell Analyzer (Merck Millipore), employing fluorescence detection at yellow–red wavelengths (576 to 680 nm) with excitation at 532 nm. Separately, for the cell cycle analysis, cells were seeded at a density of 10,000 cells per well and incubated at 37 °C in a medium enriched with 10% FBS. Post-incubation with Arylquin 1 (concentrations of 0, 1, 2.5, and 5 µM) for 72 h, the cells underwent washing with PBS and centrifugation. The pellet was then fixed in cold 70% ethanol and stored at 20 °C pending further analysis. Prior to cell cycle analysis, the cells were washed again with PBS and prepared according to the guidelines provided by the cell cycle assay manufacturer.