Clonogenic Assay

Clonogenic technique by Puck and Marcus was used for cell survival analysis. Briefly, compounds were generated as per the dose optimization section and administered into T25 flasks (Falcon). Cells were detached from stock T75 flasks (Falcon) and resuspended in medium to generate a single-cell suspension. Sample aliquot of the cell suspension was counted with the Z2 Cell Counter (Beckman Coulter) to generate values of viable cells. Following administration of compounds into flasks containing varying concentrations of each compound, cells were plated into each T25 flask (Falcon). Cell cultures were incubated for their respective clonogenic period, approximately 9 days for HCT116 p53 wt and HCT116 p53 null containing flasks and 11 days for HT29 flasks. Cells were stained following their clonogenic period of incubation with 25% carbol fuchsin in water where macroscopic colonies equal to and over 50 cells satisfy the criteria of reproductive cell survival. Total of 3 independent experiments were conducted with 3 replicates per experiment (n = 3).