3.2. In-Vitro Cytotoxicity Study (MTT Assay)

The cells suspended in the corresponding culture medium were inoculated in 96-well microtiter plates at a density of 1500–3000 cells per well, and incubated for 24 h at 37 °C in a humidified atmosphere with 95% air and 5% CO₂. Different concentrations (100, 50, 25, 12.5 and 6.25 μM) of DOX, cis-Pt or the test compounds were added in the line of cell (MDA-MB-231) in 96 h incubation. The cells were incubated for 4 h with 20 μL of 5 mg/mL MTT solution. Supernatant from each well was carefully removed, and the media were then replaced with 100 μL of dimethyl sulfoxide to dissolve the purple colored formazan crystals formed in the wells, and their absorbance were measured at 492 nm with a microplate reader (Synergy-HT, BioTek Instruments, Winooski, VT, USA); 100 μL DMSO was set as the blank control. The hypoxic condition was achieved by placing cells in a sealed hypoxia incubator chamber (Catalog Number 27310, Stemcell Technologies, Inc., Vancouver, BC, Canada) filled with 5% CO₂ and 95% N₂ [23]. For the hypoxia group, Different concentrations (100, 50, 25, 12.5 and 6.25 μM) of DOX, cis-Pt or the test compounds were added in the line of cell (MDA-MB-231) in 24 h incubation under hypoxia condition. Then the cells were moved into normoxic condition and cultured for additional 72 h.