2.2.9. Cell Cycle Studies

HCT 116 cells were cultured in 24 well-plates (250,000 cells/well) in a final DMEM volume of 500 μL. In these conditions, cells were subjected to PZQ, VHS, SEP, IP PZQ–VHSet, and IP PZQ–SEPet samples for 48 h. Increasing concentrations of each sample (0.8 μM, 4 μM, 20 μM, and 100 μM) were used. Cells entering the apoptotic or necrotic cycle were detected with a propidium iodide staining procedure, as explained in a previous protocol [40]. Briefly, once the cells were in contact with each sample, they were collected and washed with 2 mL of phosphate-buffered saline (PBS) at 4 ◦C and subsequently fixed with ethanol 70◦ (Vf = 1 mL, diluted 1:9 in PBS) for 5 min, while maintained on ice. Ethanol was withdrawn and fixed cells were washed with PBS. Then, they were resuspended in a solution consisting of 250 μL of PBS and 250 μL of a DNA extraction solution (0.2 M Na2HPO4, 0.1 M C6H8O7, pH 7.8) for 10 min at 37 ◦C. Then, the supernatant removed and 200 μL of the staining solution were added (8 μL propidium iodide (1 mg/mL) and 2 μL RNAse 100 (μg/mL)), the cells further incubated in the dark at 37 ◦C for 10 min. A FACScalibur cytometer (Becton Dickinson and Co., Franklin Lakes, NJ, USA) was used to measure the fluorescence (FL2 detector). Sub-G1 cells (dead cells, that is, cells that entered either necrotic or apoptotic phases) were detected by the Cell Quest software (Version 1.0.2, BD, Biosciences, Franklin Lakes, NJ, USA).
