Cytotoxicity Assay of Breast Cancer Cell Lines

The human breast cell lines BT549, Hs578T, MDA-MB-231, and MDA-MB-468 were obtained from American Type Culture Collection (Rockville, MD, USA). Cells were cultured in RPMI 1640 medium (Mediatech Inc., Manassas, VA, USA) for BT549 and 4T1 cells, in 1:1 DMEM/F12 (Gibco, Thermo Scientific, Waltham, MA, USA) for MDA-MB-468 cells, and in DMEM medium (Mediatech Inc., Manassas, VA, USA) for MDA-MB-231 and Hs578 cells. Culture media were supplemented with 10% fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA) and 1% penicillin/streptomycin (Gibco, Thermo Scientific, Waltham, MA, USA).
Cells (4000 per well) were plated in a 96-well plate format in 100 μL growth medium. Cells were treated with dimethyl sulfoxide (DMSO) or drugs the next day at the indicated concentrations and incubated for an additional 3 days. Viable cells were determined by the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Promega, Madison, WI, USA) [33 (link)]. After treatment, the media were removed and MTT dye was added to each well and incubated for 4 h according to the manuscripter’s instruction. The resulting formazan crystals were dissolved in DMSO after removal of the media. Absorbance was read at 570 nm. The half maximal inhibitory concentration IC₅₀ was determined using the Calcusyn software package (Biosoft, Ferguson, MO, USA).