CFI-400945

MDA-MB-468, MCF-7, HCC1954, MDA-MB-231, SKBr-3, Cal-51, and BT-20 breast cancer cells are seeded into 96-well plates at 3000, 4000, 4000, 2500, 4000, 3000, and 6000 cells per 80 μL, respectively, 24 h before compound overlay and cultured at 37℃ and 5% CO2. Compounds are prepared as 10 mM stocks in 100% DMSO. Each 10 mM stock is diluted with DMEM (Dulbecco's Modified Eagle's Medium) cell growth medium containing 10% FBS such that the final concentrations ranged from 50 nM to 250 μM. Aliquots (20 μL) from each concentration are overlaid to 80 μL of preseeded cells to achieve final concentrations of 10 nM to 50 μM. After 5 d, the cells are fixed in situ by gently removing the culture media and adding 50 μL of ice-cold 10% trichloroacetic acid (TCA) per well and incubation at 4 °C for 30 min. The plates are washed with water five times and allowed to air-dry for 5 min. Then 50 μL of 0.4% (w/v) sulforhodamine B (SRB) solution in 1% (v/v) acetic acid is added to each well,

followed by incubation for 30 min at rt. The plates are washed four times with 1% acetic acid to remove unbound SRB and air-dried for 5 min. The SRB is solubilized with 100 μL of 10 mM Tris pH 10.5 per well, and absorbance is read at 570 nm. The percentage (%) of relative inhibition of cell viability is calculated.