Cell cycle progression

Cellular DNA content was analyzed via flow cytometry to provide information on the distribution of cells in the various stages of the cell cycle after ESE-16 exposure. Propidium iodide (PI) is a fluorogenic, nuclear stain that stoichiometrically binds to nucleic acids, with fluorescence emissions directly associated to the DNA content of the cell. Therefore, the subpopulations of cells can be differentiated via fluorescence intensity due to cells having varying amounts of DNA in the assorted stages of the cell cycle [52, 53]. As PI binds to DNA, it also reacts with double-stranded RNA. RNase A was therefore added to the staining solution to digest any RNA prior to flow cytometry [54]. The phases identified were G₁, S (DNA synthesis) and G₂/M (mitotic). Cells in the sub-G₁ (apoptotic population) category were also detected due to their low DNA content because of nuclear fragmentation [52].

After seeding in 25-cm² culture flasks and allowing for cell attachment overnight, HeLa cells were exposed to 0.2 μM ESE-16 for 24 h. Following trypsinization, the cells were resuspended in 1 ml growth medium. They were then pelleted by centrifugation (300 x g) for 5 min and resuspended in 1 ml ice cold PBS. The cells were further washed and resuspended in 200 μl of ice-cold PBS containing 0.1% FCS. Ice-cold 70% ethanol (4 ml) was added in a drop wise manner and stored at 4 °C for 24 h. The following day, the cells were pelleted, the supernatant was discarded and the cells were resuspended for incubation in 1 ml of PBS containing PI (40 μg/ml), RNAse A (100 μg/ml) and triton X-100 (0.1%) at 37 °C, 5% CO₂ for 45 min. PI fluorescence was measured using a fluorescence activated cell sorting (FACS) FC500 System flow cytometer, equipped with an air-cooled argon laser (Beckman Coulter) with at least 30,000 cells per sample evaluated using CXP software (Beckman Coulter). The data from cell debris and aggregated cells were excluded from analyses. Cyflogic 1.2.1 (CyFlo) software was used to calculate cell cycle distributions by assigning relative DNA content per cell to sub-G₁, G₁, S and G₂/M fractions.