*2.2. Preparation of Solutions*

Supplemented RPMI was prepared by mixing 500 mL of phenol red-free RPMI with foetal calf serum (FCS, DKSH, Victoria, Australia) at 10% for MCF-7 and T47D, and 20% for OVCAR-3 cells, and 1% *v*/*v* of 10,000 unit/mL penicillin + 10 mg/mL streptomycin (pen-strep). Supplemented RPMI with 20% FCS also contained 5 μg/mL of recombinant human insulin for use with OVCAR-3 cells. Supplemented DMEM/F-12 was prepared by mixing phenol red-free DMEM/F-12, 10% FCS and 1% *v*/*v* of pen-strep. A total of 10 mL Hank's balanced salt solution (HBSS, provided by the DCFDA ROS assay kit manufacturer) was added to 90 mL ddH2O. DCFDA was diluted in 1X HBSS to generate a solution of 10 μM. The DCFDA ROS assay positive control, ter-butyl hydrogen peroxide (TBHP), was diluted in supplemented media (RPMI or DMEM/F12) without phenol red, to give final concentrations of 12.5 and 50 μM. Stock solutions of 100 μM Dox and 1000 μM 4-hydroperoxycyclophosphamide (4-Cyc, ThermoFisher Scientific, Victoria, Australia) were prepared in supplemented media (RPMI

or DMEM/F-12) and kept at 4 and −20 ◦C, respectively, for a maximum of three months. α and γ tocopherol were diluted in 100% dimethyl sulfoxide (DMSO) to a concentration of 1000 μM. These stock solutions were kept at 4 ◦C for a maximum of three months. Further dilutions were made using supplemented media, and the concentration of DMSO the cells were exposed to was lower than 0.8% DMSO. The crystal violet stain (0.5%) was prepared in a 50% methanol (99.9% pure). Destain solution for the crystal violet assay was prepared with 100% acetic acid diluted to 33% with demineralised water.
