5.6. AlamarBlue cell viability assays.

All compounds were evaluated for cytotoxic effect on FHC, FHs 74Int, DLD-1, HT-29, and HCT 116 cells using an AlamarBlue-based cell viability assay as per previously reported procedures.^{29, 56–60, 72, 73} Cells grown to 80% confluence were harvested and diluted in respective growth media, 1,500 cells in 45 μL were dispensed per well in 384-well plates, then plates were sealed with “Breathe Easy” oxygen permeable membranes and incubated for 24 h at 37°C and 5% CO₂. The following day, 1 μL of the compound stocks (10 mM to 4.6 μM, 3-fold dilutions in DMSO) were pre-diluted by pin-transfer into 25 μL of the respective growth media. Then, 15 μL aliquots of the diluted compounds were added to the cell assay plates to give inhibitor concentration ranges of 100 μM to 46 nM during the assays. Plates were sealed again with “Breathe Easy” oxygen permeable membranes and incubated at 37°C and 5% CO₂ for an additional 48 h for the general evaluation of all inhibitors, or for 24, 48, 72, and 96 h to establish time-course cell viability profiles for compounds 19 and 20 against HCT 116 cells. The AlamarBlue reporter reagents were then added in an amount equal to 10% of the volume in the well, the plates were incubated at 37°C and 5% CO₂, and sample fluorescence (535 nm excitation, 590 nm emission) was read using a Molecular Devices FlexStation II 384-well plate reader. Readings were taken between 4–24 h of incubation to achieve signals in the linear range of detection, typically between 30–60% conversion of resazurin to resorufin. Cell viability was calculated as per vendor instructions (ThermoFisher - AlamarBlue cell viability assay manual). EC₅₀ (half-maximal efficacy against cancerous cells) and CC₅₀ (half-maximal cytotoxicity against non-cancerous cells) values for the test compounds were obtained by plotting the % resazurin reduction results in GraphPad Prism and analyzing by non-linear regression using the variable slope dose-response equation. Results presented represent the averages of EC₅₀ and CC₅₀ values obtained from at least three independent experiments. Selectivity indices (SI) were calculated as the average of each ratio between CC₅₀ values obtained from testing against non-cancerous cells (FHC & FHs 74Int) divided by EC₅₀ values obtained from testing against HCT 116 colorectal cancer cells. For the compound 19 and 20 time-course profiles against HCT 116 cells, results were further normalized with 100% viability corresponding to the average of the % resazurin reduction across the wells containing the three lowest compound concentrations within the respective dose-response curves. Results presented represent the averages of values obtained of quadruplicates of 2 independent experiments (i.e. 8 replicates).