<b>Evaluation of the combinatory anticancer effect of chemotherapeutic compounds and Prodigiosin</b><b> against HCT116, LoVo, and A549 Cell lines (</b>Apoptose.C Cycle-PI)

After treatment with 20 µM of PG, ½ IC50 5-FU and 20 µM PG + ½ IC50 5-FU, cells were trypsinized and rinsed with PBS then fixed with 70% cold alcohol overnight. After fixation, cells were washed twice with cold PBS and incubated with 500 µl of PBS, 12µl of propidium iodide (PI) 20 mg/ml, and 2.5 µl of RNaseA (20 mg/ml) for 60 min at 37 °C in obscurity. Samples were then incubated at 4 °C until Flow Cytometry analysis. Cells were analyzed with Attune Nxt flow acoustic focusing cytometer (Thermo Fisher) equipped with a 488-argon laser. For each sample data were collected for 10.000 events, using linear amplification, at a flow rate of 100 μL/ min. The red fluorescence corresponding to the PI fluorescence signal was collected at the wavelength of 695 ± 40 nm (channel BL3). Unstained and untreated cells were used as negative and positive controls, respectively. Cell cycle distribution was determined using BL3-A peak versus counts and each assay was performed in Triplicate.