2.7. Determination of cell viability

The colloidal Pt sample was filtered through a 0.22 μM PES filter for sterilization. Vybrant® MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) Cell Proliferation Assay (Life Technologies, Carlsbad, CA, USA, Cat. No: M6494) was used for determination of cell viability of Caco-2, HCT-116 and HT-29 cell lines in the presence of different concentrations of colloidal Pt. First, 5 × 10³ cells per well were plated in 96-well plates in their corresponding growth medium. Cell proliferation was determined under the following conditions:

Treatment with colloidal Pt only (1%–20% v/v diluted in the corresponding cell culture medium) for 48 h to determine the effect of colloidal Pt on cell viability.

Treatment first with 20% colloidal Pt for 24 h then 1 h with 250 μM-5 mM H₂O₂ (Sigma-Aldrich, Cat No: 216763) after removal of Pt containing growth medium.

Co-treatment with Pt (20%) and H₂O₂ or co-treatment with Pt (20%) and 5 mM-80 mM 2, 2-azobis (2-amidinopropane) dihydrochloride (AAPH, dissolved in dH₂O, Sigma-Aldrich, Cat. No: 440914) for 1 h.

Following the treatments, the medium was removed and the MTT cell viability assay was carried out according to the manufacturer's instructions. Briefly, 12 mM MTT stock solution was diluted with growth medium in 1:10 ratio and distributed to each well as 100 μl. Empty wells containing only 100 μl of diluted MTT were used as blank. The plates were incubated at 37 °C for 4 h. In order to dissolve the formazan crystals, 100 μl of 1/10 (g/mL) SDS/dH₂O (containing 0. 01 N HCl) solution was added for each well followed by incubation at 37 °C for 16 h. Finally, measurements were taken at 570 nm in MultiSkan GO Microplate Spectrophotometer (Thermo Scientific) microplate reader. Sterile dH₂O was used as vehicle in colloidal Pt and AAPH treatments. Experiments were carried out with at least two biological replicates each with eight technical replicates.